Registration Dossier

Administrative data

Description of key information

Testing of d-limonene in rabbits demonstrated some moderate irritating effects on skin and slight irritating effects on eyes.

These irritation effects were not sufficiently severe for classification of the substance as skin or eye irritant according to CLP criteria.

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:
June 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD guideline 404 with minor deviations: no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Species:
rabbit
Strain:
other: albino
Details on test animals and environmental conditions:
None
Type of coverage:
semiocclusive
Preparation of test site:
other: intact
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
7 days
Number of animals:
Three
Details on study design:
SCORING SYSTEM: Draize scoring system
- tested in a multi-patch study
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
2
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1.33
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritant / corrosive response data:
- Desquamation from skin surface was observed in all animals on Day 7
- See table 1 for individual animal scores
Other effects:
No data

Table 1: Skin irritation data for individual rabbits

 

Rabbits No.

1*

2**

3*

Erythema

1 hour

2

2

1

24 hours

2

2

2

48 hours

2

2

2

72 hours

2

2

2

Day 7

1

2

1

Mean (24 hours + 48 hours + 72 hours)

2

2

2

Oedema

1 hour

3

4

3

24 hours

2

2

2

48 hours

1

2

1

72 hours

1

2

1

Day 7

1

3

1

Mean (24 hours + 48 hours + 72 hours)

1.33

2

1.33

* marked desquamation from skin surface after 7 days

** desquamation from skin surface after 7 days

Interpretation of results:
not irritating
Conclusions:
d-Limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In a primary dermal irritation study performed according to OECD Guideline 404 and in compliance with GLP, three albino rabbits were dermally exposed to 0.5 mL of undiluted d-limonene, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 2, 2, 2 for erythema score and 1.33, 2, 1.33 for oedema score. Signs of erythema and oedema, as well as desquamation from skin surface, were observed in all animals on Day 7. In this study, d-limonene was found to be skin irritant when applied topically to the rabbit.

 

Therefore, d-limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No 1272/2008.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD guideline 404 with minor deviations: no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
no details on test animals and environmental conditions; no details on patch material; observations not followed until the reversibility of the effects (only 7 days)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Species:
rabbit
Strain:
other: albino
Details on test animals and environmental conditions:
None
Type of coverage:
semiocclusive
Preparation of test site:
other: intact
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
7 days
Number of animals:
Four
Details on study design:
SCORING SYSTEM: Draize scoring system
- tested in a multi-patch study
Irritation parameter:
erythema score
Basis:
mean
Remarks:
all animals
Time point:
other: 24, 48 and 72 h
Score:
1.92
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritation parameter:
edema score
Basis:
mean
Remarks:
all animals
Time point:
other: 24, 48 and 72 h
Score:
1.33
Max. score:
4
Reversibility:
not fully reversible within: 7 days
Irritant / corrosive response data:
- Slight desquamation from skin surface was observed in 2/4 animals on Day 7
- See table 1 for individual animal scores
Other effects:
No data

Table 1: Skin irritation data for individual rabbits

 

Rabbits No.

#1*

#2

#3

#4*

Erythema

1 hour

1

1

0

2

24 hours

2

2

1

2

48 hours

2

2

2

2

72 hours

2

2

2

2

Day 7

2

1

2

2

Mean (24 hours + 48 hours + 72 hours)

2

2

1.67

2

Oedema

1 hour

4

3

3

3

24 hours

2

1

2

2

48 hours

1

1

1

2

72 hours

1

0

1

2

Day 7

1

0

2

2

Mean (24 hours + 48 hours + 72 hours)

1.33

0.67

1.33

2

* slight desquamation from skin surface after 7 days

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
d-Limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In a primary dermal irritation study performed according to OECD Guideline 404 and in compliance with GLP, four albino rabbits were dermally exposed to 0.5 mL of undiluted d-limonene, under a semi-occlusive patch for 4 hours to intact skin of the flank. Animals were then observed for 7 days. Irritation was scored by the method of Draize at 1, 24, 48 and 72 hours and 7 days after exposure.

 

Mean individual scores at 24, 48 and 72 h after exposure for the 4 animals were 2, 2, 1.67, 2 for erythema score and 1.33, 0.67, 1.33, 2 for oedema score. Slight desquamation was observed in 2/4 animals on day 7. In this study, d-limonene was found to be skin irritant when applied topically to the rabbit.

 

Therefore, d-limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No 1272/2008.

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
L-Limonene is the S-isomer of d-limonene. Therefore, data on l-limonene can be used for extrapolation to d-limonene. See read-across justification document in section 13.
Reason / purpose:
read-across source
Remarks on result:
other: moderate redness: 3/10 animals; moderate edema: 6/10 animals
Other effects:
No data

None

Conclusions:
Topical application of l-limonene resulted in moderate redness and moderate edema in 3/10 and 6/10 animals, respectively.
Executive summary:

In a primary dermal irritation study, ten rabbits were dermally exposed to l-limonene.

 

Under the test conditions, moderate redness and moderate edema were observed in 3/10 and 6/10 animals, respectively.

Based on the results of this study, no conclusion regarding the classification of l-limonene can be reached according to CLP regulation (EC) No 1272/2008 or GHS.

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:
From May 10 to 21, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in compliance with OECD guideline 405 with minor deviation: no data on tool used to asses score
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
no data on tool used to asses score
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
no data on tool used to asses score
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Elevage de Gerome, Quartier Labaste, France
- Age at study initiation: 11 or 13 weeks
- Weight at study initiation: 2.04-3.05 kg
- Housing: Each animal housed in individual box
- Diet (e.g. ad libitum): Foodstuff (SDS - C15); ad libitum
- Water (e.g. ad libitum): Tap water (public distribution system); ad libitum
- Acclimation period: Five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
- Humidity (%): 30-70%
- Air changes (per hour):15/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume with unit): 0.1 mL
Duration of treatment / exposure:
No washing was done
Observation period (in vivo):
Seven days
Number of animals or in vitro replicates:
Three males
Details on study design:
SCORING SYSTEM:

Chemosis
No swelling …0
Slight swelling, including the nictitating membrane…1
Swelling with eversion of the eyelid...2
Swelling with eyelid half-closed…3
Swelling with eyelid more than half-closed…4

Discharge
No discharge…0
Slight discharge (normal slight secretion in the inner corner not to be taken into account)…1
Discharge with moistening of the eyelid and neighboring hairs…2
Discharge with moistening of the eyelids and large areas around the eye…3

Redness
Blood vessel normal…0
Vessels significantly more prominent than normal…1
Vessels individually distinguishably with difficulty
Generally red coloration….2
Generally deep red coloration….3

Iris
Normal…0
Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly…1
No reaction to light, haemorrhage, significant damage (any or all of these characteristics)…2

Cornea: degree of opacity
No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)…0
Translucent areas (diffuse or disseminated), iris details clearly visible…1
Early identifiable translucent area, iris details slightly obscured…2
Opalescent area, no iris details visible, pupil outline scarcely distinguishable…3
Total corneal opacity, completely obscuring the iris and pupil…4

Cornea: extent of opacity
Opaque area present but covering one quarter or less…1
Between one quarter and half…2
Between half and three quarters…3
Between three quarters and the entire surface…4
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0.3
Max. score:
3
Reversibility:
fully reversible within: 2 days
Irritation parameter:
iris score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0.3
Max. score:
4
Reversibility:
fully reversible within: 2 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1
Max. score:
4
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
1.3
Max. score:
3
Reversibility:
fully reversible within: 4 days
Irritation parameter:
iris score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
2
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Remarks:
mean individual score
Time point:
24/48/72 h
Score:
0
Max. score:
4
Reversibility:
other: not applicable
Irritant / corrosive response data:
- Slight to moderate redness of conjunctivae associated with moderate chemosis was noted within 1 hour of instillation; irritation completely resolved within 7 days.
- See table 1 for more details
Other effects:
No data

Table 1: Individual and mean scores of conjunctivae, iris and cornea

 

Animal

Time after treatment

Conjunctivae

Iris

Cornea 

Chemosis

Redness

Lesion

Opacity

1

1 hour

2

1

0

0

24 hours

1

1

0

0

48 hours

1

0

0

0

72 hours

1

0

0

0

Day 4

1

0

0

0

Day 7

0

0

0

0

Mean (24, 48 and 72 hours)

1

0.3

0

0

2

1 hour

2

2

0

0

24 hours

1

1

0

0

48 hours

0

1

0

0

72 hours

0

1

0

0

Day 4

0

0

0

0

Mean (24, 48 and 72 hours)

0.3

1

0

0

3

1 hour

2

2

0

0

24 hours

2

2

0

0

48 hours

1

1

0

0

72 hours

0

1

0

0

Day 4

0

0

0

0

Mean (24, 48 and 72 hours)

1

1.3

0

0

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
d-Limonene is not classified as irritating to the eye according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an eye irritation study conducted according to the OECD Guideline 405 and in compliance with GLP, three male rabbits of the New Zealand White strain were exposed to 0.1 mL of undiluted d-limonene in one of the eyes while the other eye corresponded to the control. The eyes were examined for irritation scores at 1 hour and 1, 2, 3, 4 and 7 days after dosing.

 

Instillation of d-limonene resulted in slight to moderate redness of conjunctivae associated with moderate chemosis in all treated animals after 1 hour of instillation. The irritation completely resolved within 7 days. Mean individual scores at 24, 48 and 72 hours after exposure for the 3 animals were 0, 0, 0 for cornea score; 0, 0, 0 for iris score; 0.3, 1, 1.3 for conjunctivae score and 1, 0.3, 1 for chemosis score.

 

Therefore, d-limonene is not classified as irritating to the eyes according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a primary dermal irritation study performed according to OECD Guideline 404 and in compliance with GLP, three albino rabbits were dermally exposed to d-limonene, under a semi-occlusive patch for 4 hours.

Mean individual scores at 24, 48 and 72 h after exposure for the 3 animals were 2, 2, 2 for erythema score and 1.33, 2, 1.33 for oedema score. Signs of erythema and oedema, as well as desquamation from skin surface, were observed in all animals on Day 7.

This test was repeated under the same conditions. In this second experiment: mean individual scores at 24, 48 and 72 h after exposure for 4 animals were 2, 2, 1.67, 2 for erythema score and 1.33, 0.67, 1.33, 2 for oedema score. Slight desquamation was observed in 2/4 animals on day 7.

One suporting study was available: in a study performed with l-limonene (in vivo testing for skin irritation with no detailed information and no scoring system), moderate redness and moderate edema was observed in 3/10 and 6/10 animals, respectively.

In an eye irritation study conducted according to OECD Guideline 405, three male rabbits of the New Zealand White strain were exposed to d-limonene in one of the eyes while the other eye corresponded to the control.  

Instillation of d-limonene resulted in slight to moderate redness of conjunctivae. Mean individual scores at 24, 48 and 72 hours after exposure for the 3 animals were 0, 0, 0 for cornea score; 0, 0, 0 for iris score; 0.3, 1, 1.3 for conjunctivae score and 1, 0.3, 1 for chemosis score.

 

Justification for classification or non-classification

According to CLP, the major criterion for the irritant category is that at least 2 of 3 tested animals have a mean score of ≥ 2.3 - ≤ 4.0. Under the test conditions, d-limonene is classified as ‘R38 Irritating to skin’ according to Directive 67/548/EEC but it is not classified according to CLP Regulation (EC) No 1272 /2008, as the maximum mean value in all animals was 2 for skin erythema. However, only slight signs of reversibility are observed at Day 7 and the current harmonised classification classifies the substance as irritating to skin. Therefore the substance is classified in category 2 for skin irritation according to CLP Regulation (EC) No 1272 /2008 in order to stay in line with the current harmonised classification.

As only slight to moderate redness of conjunctivae (mean score < 2.0) was observed following rabbit testing for eye irritation, d-limonene is not classified as irritating to the eyes according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.