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Diss Factsheets

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.
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.


D-Limonene is one of the main constituent of REACTION MASS OF 1,4-CINEOLE, 1,8-CINEOLE, DL-LIMONENE AND TERPINOLENE so data on D-limonene can be extrapolated to the substance multiconstituent

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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
D-Limonene is one of the main constituent of REACTION MASS OF 1,4-CINEOLE, 1,8-CINEOLE, DL-LIMONENE AND TERPINOLENE so data on D-limonene can be extrapolated to the substance multiconstituent
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
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. 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.

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:
D-Limonene is one of the main constituent of REACTION MASS OF 1,4-CINEOLE, 1,8-CINEOLE, DL-LIMONENE AND TERPINOLENE so data on D-limonene can be extrapolated to the substance multiconstituent
Reason / purpose for cross-reference:
read-across source
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.


 


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. 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.

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

Eye irritation

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.