Registration Dossier

Administrative data

Description of key information

Skin irritation:

In the key study of Park (2007) performed according to the Korean Food and Drug Administration (KFDA) guidelines (equivalent or similar to OECD TG 403), the skin irritation potential was investigated in New Zealand white rabbits using repellent solution prepared of 0.5 g (2.0%) of alpha terpinene.

The local skin reactions were read and recorded at 24 h and 72 h using the scoring system. A skin biopsy was taken at 72 h after treatment for histological studies. The skin biopsies were fixed in 10% buffered formalin solution for several days, then underwent ethanol series dehydration and embedded in paraffin. Sections were stained with hematoxylin-eosin and examined under a light microscope.

Results demonstrated, that no acute abnormalities were observed to intact or abraded skin after application of 2% alpha terpinene in rabbits.

In other study of Kitahara (1993), alpha terpinene was assessed for the skin irritation potential in rats. Gel ointments containing 2 g of alpha terpinene were applied to the male rat abdominal skin for 10 hr. After 10 hr, the study was terminated and the histopathological endpoints including epidermis, dermis, hypodermis and skin appendages and irritation scores were evaluated. Histopathological findings indicated that alpha terpinene caused a moderate skin irritation in rats. The moderate liquefaction and desquamation of the epidermis were noticed (irritation score 3). Marked collagen swelling in hypodermis (irritation score 4) with very slight focal haemorrhage and moderate oedema (irritation score 3) were demonstrated, as well.

Conclusion for not classification:

The study of Kitahara (1993) did not follow any recognised testing guideline. The exposure to alpha terpinene was 2.5 times longer than it is recommended in the OECD TG 404 (application of a

test substance for up to 4 hours).Furthermore,the albino rabbit is the preferable laboratory animal for skin irritation testing.Also, the reversibility of effects could not be evaluated as the study was terminated after the exposure period of 10 hr. In addition,

It is therefore concluded that results obtained in the key study of Park (2007) give the reliable prediction for skin irritation potential of alpha terpinene.

 

Eye irritation:

In the key study of Park (2007), the primary eye irritation according to the “Guidelines for Toxicity Studies of Drugs” provided by Korea Food and Drug Administration (KFDA), was performed. The corneal opacity, iris and conjunctival redness, oedemas were recorded and classified according to the guideline of KFDA. For the purpose of this registration, the scores obtained based on KFDA guideline were compared to the scoring system of the OECD TG 405 - grading of ocular lesions.

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 mean grades of ocular reaction (conjunctivae, cornea and iris) recorded after 24, 48 and 72 hours were calculated, and the mean scores were approx. 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin 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:
data from handbook or collection of data
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
according to guideline
Guideline:
other: Korean Food and Drug Administration (KFDA) guidelines
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: alpha terpinene was purchased from Sigma (St. Louis, MO)

FORM AS APPLIED IN THE TEST: Formulation of test repellents.
Repellent solution prepared using 0.5 g (2.0%) of alpha terpinene.

Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system 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


Type of coverage:
occlusive
Preparation of test site:
shaved
Remarks:
dorsal trunk
Vehicle:
other: Formulation of test repellents
Controls:
not specified
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): A volume of 0.5 mL repellent solution
- Concentration: 0.5 g (2.0%) of alpha terpinene in repellent solution

NEGATIVE CONTRL - yes

POSITIVE CONTROL - no
Duration of treatment / exposure:
4 hrours
Observation period:
24 h and 72 h
Number of animals:
Three males and three females were tested
Details on study design:
TEST SITE
- Area of exposure: dorsal trunk
- % coverage: 100% After applying the test compound polyethylene wrap was secured around the trunk of the rabbit. Rabbits were placed in a restraining cage for 4 h treatment. After that time which wraps were removed.
- Type of wrap if used: 1-inch square gauze patch was placed on the skin and secured with adhesive tape.

OBSERVATION TIME POINTS - 24 h and 72 h

SCORING SYSTEM: provided

Four different skin sensitivity cohorts were examined, as follows:
(1) non-treated at intact site (NTN group)
(2) non-treated at abraded site (NTA group),
(3) 2% a -terpinene solution at intact site (2TN group),
(4) 2% a -terpinene solution at abraded site (2TA group).

Each test rabbit had 3 intact sites and 3 abraded sites.
Irritation parameter:
erythema score
Remarks:
scoring based on the final results
Basis:
mean
Time point:
24/48/72 h
Score:
> 0 - <= 2
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
other: no scoring provided
Remarks:
No acute abnormalities were observed to intact or abraded skin after application of 2% alpha terpinene in rabbits.
Irritation parameter:
edema score
Remarks:
scoring based on the final results
Basis:
mean
Time point:
24/48/72 h
Score:
> 0 - <= 2
Max. score:
4
Reversibility:
fully reversible
Remarks on result:
other: no scoring provided
Remarks:
No acute abnormalities were observed to intact or abraded skin after application of 2% alpha terpinene in rabbits.
Interpretation of results:
other: not skin irritant
Conclusions:
No acute abnormalities were observed to intact or abraded skin after application of 2% alpha terpinene in rabbits.




Executive summary:

In this study, the skin irritation potential was investigated in New Zealand white rabbits using the repellent solution containing 0.5 g (2.0%) of alpha terpinene.

The local skin reactions were read and recorded at 24 h and 72 h using the scoring system. A skin biopsy was taken at 72 h after treatment for histological studies. The skin biopsies were fixed in 10% buffered formalin solution for several days, then underwent ethanol series dehydration and embedded in paraffin. Sections were stained with hematoxylin-eosin and examined under a light microscope.

Results demonstrated, that no acute abnormalities were observed to intact or abraded skin after application of 2% alpha terpinene in rabbits.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test:
Investigation of the dermal irritancy potential of the test material, alpha terpinene by evaluation of histopathological findings of rat abdominal skin at 10 hr after application of gel ointments containing alpha terpinene.

- Short description of test conditions:
Gel ointments containing the test material dissolved in ethanol were prepared,
and placed in 16-mm glass cells. The glass cells were attached to the shaved skin with adhesives, and the ointments were removed after 10 hours. A 1-cm2 sample of skin was taken from 2 of the dosing sites for each preparation, and the excised skin was fixed in 10% neutral carbonate-buffered formalin for at least 24 hours before being processed.

- Parameters analysed / observed:
Sections of the skin were dehydrated, embedded in paraffin wax, and stained for examination by light microscopy.

GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: alpha terpinene, Tokyo Chemical Industries CO.,Ltd.

FORM AS APPLIED IN THE TEST - The gel ointments were prepared, as follows: alpha terpinene was dissolved in ethanol. Separately, carboxyvinyl polymer and triethanolamine were dissolved in distilled water. Both compounds were then mixed well, and the resulting gel ointment was stored at room temperature for 24 h under air-tight conditions prior to use.


Species:
rat
Strain:
Wistar
Remarks:
males
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Male rats
- Weight at study initiation: 160-190 g
Type of coverage:
other: Application of gel ointments
Preparation of test site:
shaved
Remarks:
rat abdominal skin
Vehicle:
other: ethanol
Remarks:
0.5 (v/v) %
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
- Concentration: 0.1 % (w/v)

VEHICLE
- Amount(s) applied ethanol 0.5 (v/v) %

NEGATIVE CONTROL - untreated


Duration of treatment / exposure:
10 hrs
Observation period:
10 hr after exposure
Number of animals:
not specified
Details on study design:
TEST SITE:
After anesthetization with a urethane saline solution (25%, 3 ml/kg i.p.), the rats were secured on their backs and the hair on the abdominal skin was removed with an electric animal clipper. Glass cells (16 mm inner diameter, IO mm height) containing the gel ointment under test (1.5 g) were attached to the shaved skin with cyanoacrylate-type adhesives After IO h, the gel ointments were removed. Two of the dosing sites on the skin for each preparation were immediately taken from the rats (each sample was about I cm2 of area). The excised skin was fixed in 10% neutral carbonate-buffered formalin for at least 24 h before routine procession and then cut vertically against the skin surface at the central region in 4 mm widths. Each section was dehydrated using a graded series of ethanol solutions and was then embedded in paraffin wax. Tissues were divided into small pieces (about 3 µm in thickness) and stained with hematoxylin and eosin. All sections were examined by an optiphoto light microscope (Optiphoto, Nikon).

SCORING SYSTEM:

The irritaiton score system was applied, as follows:

0 - no change
1 - very slight
2 - slight
3 - moderate
4 - marked
Irritation parameter:
edema score
Remarks:
based on 3-4 determinations
Basis:
mean
Time point:
other: 10 hrs
Score:
ca. 3
Reversibility:
not specified
Remarks on result:
probability of moderate irritation
Irritation parameter:
overall irritation score
Remarks:
based on 3-4 determinations
Basis:
mean
Time point:
other: 10 hrs
Score:
> 1 - < 4
Max. score:
4
Reversibility:
not specified
Remarks on result:
probability of moderate irritation
Interpretation of results:
other: skin irritant
Conclusions:
Based on the results of this study, alpha terpinene was demonstrated to be a skin irritant.
Executive summary:

This study was performed to assess the skin irritation effects of alpha terpinene in rat abdominal skin.

 

Briefly, gel ointments containing 2 g of alpha terpinene were applied to the male rat abdominal skin for 10 hr. After that time, the study was terminated and the irritation scores were given following the histopathological findings evaluation of epidermis, dermis, hypodermis and skin appendages.

 

Histopathological findings indicated that alpha terpinene caused a moderate skin irritation in rats. The moderate liquefaction and desquamation of the epidermis were noticed (irritation score 3). Marked collagen swelling in hypodermis (irritation score 4) with very slight focal haemorrhage and moderate oedema (irritation score 3) were demonstrated, as well.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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
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.
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
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.
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
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
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
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
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
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
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
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Justification for classification or non-classification

The available data on skin irritation of the test substance do not meet the criteria for classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.

However, based on the data on eye irritation, alpha terpinene should be classified as an eye irritant, cat. 2 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.