Registration Dossier

Administrative data

Description of key information

In in vivo studies conducted according to or similarly to OECD guideline 404 on d-limonene in rabbits, reversible signs of skin irritation were observed.

In an in vitro study conducted according to OECD guideline 492 with the registered substance on Reconstructed Human Cornea-like Epithelium, tissue viability was 82.94%.

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 constituents of multiconstituent substance REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. Therefore, data on d-limonene can be used for extrapolation to REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. See read-across justification document in section 13.
Reason / purpose:
read-across source
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
mean individual score
Time point:
other: 24, 48 and 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:
other: 24, 48 and 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:
other: 24, 48 and 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:
other: 24, 48 and 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:
other: 24, 48 and 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:
other: 24, 48 and 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
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, 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:
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 constituents of multiconstituent substance REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. Therefore, data on d-limonene can be used for extrapolation to REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE. See read-across justification document in section 13.
Reason / purpose:
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.

 

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 conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Strain:
other: reconstructed human Cornea-like Epithelium
Details on test animals or tissues and environmental conditions:
EpiOcular TM tissue Model OCL-212 supplied by MatTek
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE was applied as supplied at the dose of 50 µL
Duration of treatment / exposure:
30 minutes
Duration of post- treatment incubation (in vitro):
A 12-minute post exposure immersion period at room temperature and 1 hour and 54 minutes post exposure incubation at standard culture conditions.
Number of animals or in vitro replicates:
2 replicates.
Irritation parameter:
other: percent tissue viability
Value:
82.94
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
percent tissue viability = 31.99%
Irritant / corrosive response data:
REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE IS CONSIDERED AS NON IRRITANT AND NON CORROSIVE
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions adopted and in accordance with the Regulation EC No. 1272/2008, the test item REACTION MASS OF BETA-PHELLANDRENE AND D-LIMONENE AND L-LIMONENE does not require classification for eye irritation or serious eye damage. Also, the test item does not require classification for eye irritation or serious eye damage according to UN GHS.
No hazard statement and no signal word are required.
Executive summary:

In a GLP study conducted according to OECD 492 guideline, the irritant potential of reaction mass of beta-phellandrene and D-limonene and L-limonene was evaluated. The method is based on viability of Reconstructed Human Cornea-like Epithelium-(RhCE) when exposed to 50 µL the test substance for 30 min. The mean percent tissue viability of the RhCE replicates treated with the test item reaction mass of beta-phellandrene and D-limonene and L-limonene was 82.94% versus 31.99% in the positive control (Methyl acetate). In conclusion, under experimental conditions adopted and in accordance with the Regulation EC N° 1272/2008, the test item does not require classification as eye irritant or serous eye damage according to UN GHS. No hazard statement or signal word are required.

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.

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 were observed in 3/10 and 6/10 animals, respectively.

In a GLP study conducted according to OECD 492 guideline, the irritant potential of reaction mass of beta-phellandrene and D-limonene and L-limonene was evaluated. The method is based on viability of Reconstructed Human Cornea-like Epithelium-(RhCE) when exposed to 50 µL the test substance for 30 min. The mean percent tissue viability of the RhCE replicates treated with the test item reaction mass of beta-phellandrene and D-limonene and L-limonene was 82.94% versus 31.99% in the positive control (Methyl acetate). In conclusion, under experimental conditions adopted and in accordance with the Regulation EC N° 1272/2008, the test item does not require classification as eye irritant or serious eye damage.

Justification for classification or non-classification

An harmonised classification is available for d-limonene and it is classified as skin irritant. Therefore, as the registered substance contains more than 10% of dl-limonene, it should be classified as skin irritant according to the rules of classification for mixtures of CLP Regulation (EC) No 1272/2008.

In a GLP study conducted according to Guideline OECD 492, exposure of epithelia to the registered substance led to percent tissue viability higher than 60% therefore under experimental conditions adopted and in accordance with CLP Regulation EC No 1272/2008, the registered substance does not require classification as eye irritant or serious eye damage.