Registration Dossier

Administrative data

Description of key information

Skin irritation (WoE based on two OECD TG 404 studies): irritating

Eye irritation (WoE based on two OECD TG 405 studies): irritating

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:
weight of evidence
Study period:
27-09-1988 to 4-10-1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study was conducted according to a method similar to OECD guideline 404 and under GLP conditions.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Qualifier:
according to
Guideline:
other: EEC Commission Directive 25 april 1984, Document L251, p106-108
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Goreside Rabbits, Northchurch, Berkhamsted, Herfordshire
- Age at study initiation: 10-12 weeks
- Weight at study initiation: approximately 2 kg
- Housing:Individually housed in grid bottomed metal cages
- Diet (e.g. ad libitum): antibiotic free rabbit diet (SQC standard rabbit pellets produced by Special Diets Services, Witham, Essex), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22
- Humidity (%): 57-69
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 ml
Duration of treatment / exposure:
4 hours
Observation period:
7 days (scoring at 1, 24, 48 and 72 hours and 7 days after dosing)
Number of animals:
4 rabbits
Details on study design:
TEST SITE
- Area of exposure: approximately 2.5 cm2 of the dorsal skin per patch
- Type of wrap if used: surgical lint, held in contact with the skin under a semi-occlusive dressing

REMOVAL OF TEST SUBSTANCE
- Washing (if done): gentle swabbing with cotton wool soaked in warm water
- Time after start of exposure: 4 hrs

SCORING SYSTEM:
- Irritation was assessed and allocated a numerical value based on a grading scale (with scores 0-4).
- The system described in the Official Journal European Communities L257 (published 16th sept 1983) was used to classify the materials.
- The scoring system is in accordance with OECD guideline 404
Irritation parameter:
erythema score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #3
Remarks:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
4
Irritation parameter:
erythema score
Basis:
animal #4
Remarks:
mean
Time point:
24/48/72 h
Score:
2
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
mean
Time point:
24/48/72 h
Score:
2.3
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
mean
Time point:
24/48/72 h
Score:
2.5
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
mean
Time point:
24/48/72 h
Score:
1.3
Max. score:
4
Irritation parameter:
edema score
Basis:
animal #4
Remarks:
mean
Time point:
24/48/72 h
Score:
1.2
Max. score:
4
Irritant / corrosive response data:
One hour after the dosing period well-defined erythema was observed in two animals and very slight to well-defined erythema was apparent in two animals. Slight to moderate edema was noted in two animals at 1 hour, one rabbit exhibited moderate edema and the remaining animals exhibited moderate to severe edema.
Other effects:
Desquamation from the surface of the treated skin was observed in three animals.
Interpretation of results:
other: not irritating
Remarks:
in accordance with Annex I of 1272/2008/EC (CLP)
Conclusions:
It was concluded in the study that the substance needs to be classified according to the system described in the Official Journal of European Communities No. L257 (16 September 1983) and classification is also warranted according to the criteria outlined in Annex VI of 67/548/EEC (DSD). However, according to the current criteria for skin irritation/corrosion as outlined in Annex I of 1272/2008/EC (CLP) the substance does not need to be classified anymore for skin irritation/corrosion.
Executive summary:

In an acute dermal irritation study (similar to OECD 404, testing material, Litsea Cubeba oil, was applied at a volume of 0.5 ml over an area of 2.5 cm2 on the dorsal skin, clipped free of fur, of four rabbits. The materials were held in contact with the skin under a semi-occlusive dressing for a four hour period after which the patches were removed. Skin reaction to the material was assessed after 1, 24, 48 and 72 hours and 7 days after the dosing period.

One hour after the dosing period well-defined erythema was observed in two animals and very slight to well-defined erythema was apparent in two animals. Slight to moderate edema was noted in two animals at 1 hour, one rabbit exhibited moderate edema and the remaining animals exhibited moderate to severe edema. In general this response slowly declined. Some differences in erythema and edema reaction between animals were observed. The average scores for erythema at 24, 48 and 72 hour after dosing were 2.0 in all four animals whereas the average scores for edema at 24, 48 and 72 hour after dosing were 1.2, 1.3, 2.3 and 2.5.

It was concluded in the study that the substance needs to be classified according to the system described in the Official Journal of European Communities No. L257 (16 September 1983) and classification is also warranted according to the criteria outlined in Annex VI of 67/548/EEC (DSD). However, according to the current criteria for skin irritation/corrosion as outlined in Annex I of 1272/2008/EC (CLP) the substance does not need to be classified anymore for skin irritation/corrosion.

Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
29 November 1976 - 29 December 1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test was conducted according to methods similar to OECD 404 (performed pre-GLP), but with some deviations that do not alter the reliability.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
evaluation only up to 72 hours after treatment, erythema was not scored at 24 hours because the test sample stained the skin yellow and no OECD scoring system was used
GLP compliance:
no
Remarks:
performed pre-GLP
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9-12 weeks
Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.5 mL
Duration of treatment / exposure:
4 hours
Observation period:
72 hours
Number of animals:
10
Details on study design:
TEST SITE
- Area of exposure: Dorsal area
- Coverage: 1 inch x 1 inch = 6.5 cm2
- Type of wrap if used: gauze pad heat-sealed on to polythene sheeting, covered by adhesive tape

REMOVAL OF TEST SUBSTANCE
- Washing: Site wiped clean of excess material
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Total irritation score per animal, mean irritaton score per site and mean irritation score per site per day are calculated.

An 8-point scale was used in the study report:
- 'a' (marginal/very slight); score = 1
- 'b' (slight); score = 2
- 'c' (fairly distinct); score = 3
- 'd' (quite distinct); score = 4
- 'e'(becoming well-developed); score = 6
- 'f' (well-developed); score = 8
- 'g' (becoming severe); score = 10
- 'h' (severe); score = 12

For comparison with current CLP classification criteria for skin irritation/corrosion the scoring of the oedema formation data at 24, 48 and 72 hours was converted to the OECD scoring scale as follows by the evaluator:
- grade a (marginal/very slight) was scored as 1 according to the OECD scale
- grades b/c (slight/fairly distinct) were scored as 2 according to the OECD scale
- grades d to g (quite distinct - becoming severe) was scored as 3 according to the OECD scale
- grade h (severe) was scored as 4 according to the OECD scale
Irritation parameter:
erythema score
Remarks:
No data
Basis:
mean
Remarks:
No data available
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
other: 24 hour timepoint is lacking (due to staining of the skin by the test sample).
Irritation parameter:
edema score
Basis:
animal #1
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2.3
Irritation parameter:
edema score
Basis:
animal #2
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2.7
Irritation parameter:
edema score
Basis:
animal #3
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2.3
Irritation parameter:
edema score
Basis:
animal #4
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
3
Irritation parameter:
edema score
Basis:
animal #5
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
3
Irritation parameter:
edema score
Basis:
animal #6
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2.7
Irritation parameter:
edema score
Basis:
animal: # 7
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2
Irritation parameter:
edema score
Basis:
animal: # 8
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2.7
Irritation parameter:
edema score
Basis:
animal: # 9
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2.7
Irritation parameter:
edema score
Basis:
animal: #10
Remarks:
Mean value for oedema formation
Time point:
24/48/72 h
Score:
2.3
Irritant / corrosive response data:
- Oedema was quite distinct to well-developed (score 4-8) at 4 hours after exposure and slight to becoming well-developed (score 2-6) at 24 hours. At 48 hours and 72 hours slight to well-developed (score 2-8) oedema was observed.
- Erythema was not scored at 4 and 24 hours, as the test sample stained the skin yellow. At 48 hours fairly distinct to well-developed (score 3-8) erythema was noted, while it was classified as quite distinct to becoming well-developed (score 4-6) at 72 hours.
- Cracking and scaling was not observed at 4 hours. Cracking was marginal/very slight to slight (score 1-2) at 24 hours, while scaling was not observed. Marginal/very slight to fairly distinct (score 1-3) cracking and scaling was observed at 48 hours and at 72 hours this was classified as marginal/very slight to quite distinct (score 1-4).

The erythema data in this study could not be used for CLP-comparison as the 24 hour timepoint is lacking (due to staining of the skin by the test sample).

In the table below, the mean values for oedema formation (at 24, 48 and 72 hours) per animal are shown, based on the conversion as described in the materials and methods section.

 Animal No.  Mean value for oedema formation
 1  2.3
 2  2.7
 3  2.3
 4  3
 5  3
 6  2.7
 7  2
 8  2.7
 9  2.7
 10  2.3

9 out of 10 animals showed a mean score for oedema formation of ≥2.3.

Interpretation of results:
other: Irritating
Remarks:
in accordance with the criteria outlined Annex I of 1272/2008/EC (CLP).
Conclusions:
Under the conditions of this study, all animals developed distinct to well-defined skin irritation after treatment with undiluted Litsea cubeba for 4 hours. Translation of the results to a scoring system in line with the CLP criteria showed that 9 out of 10 animals had an oedema score of ≥2.3. Based on these findings, the test substance was considered to be irritating in accordance with the criteria outlined Annex I of 1272/2008/EC (CLP).
Executive summary:

The skin irritating potential of Litsea cubeba essential oil as tested in this study according to a method similar to OECD 404 (performed pre-GLP). Major deviation was that no OECD scoring system was used to evaluate the erythema and oedema formation. Ten rabbits were exposed for 4 hours to the test substance (semi-occlusive) and skin reactions were qualitatively evaluated for 72 hours.

Oedema was quite distinct to well-developed at 4 hours after exposure and slight to becoming well-developed at 24 hours. At 72 hours slight to becoming well-developed oedema was observed. Erythema was not scored at 4 and 24 hours, as the test sample stained the skin yellow. At 48 hours fairly distinct to well-developed erythema was noted, while it was classified as quite distinct to becoming well-dev eloped at 72 hours. Cracking and scaling was not observed at 4 hours, marginal/very slight at 24 hours, marginal/very slight to fairly distinct at 48 hours and marginal/very slight to quite distinct at 72 hours. The qualitative results for oedema were translated to the OECD scoring system by the evaluator to be able to test them against the current classification criteria. It was however not possible to derive an erythema score due to the lack of observations at one of the required timepoints.

Under the conditions of this study, all animals developed distinct to well-defined skin irritation after treatment with undiluted Litsea cubeba essential oil for 4 hours. Translation of the results to a scoring system in line with the CLP criteria showed that 9 out of 10 animals had an oedema score of ≥2.3. Based on these findings, the test substance was considered to be irritating in accordance with the criteria outlined Annex I of 1272/2008/EC (CLP).

Endpoint:
skin irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
9 May 1977 - 31 May 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to methods similar to OECD guideline 405 and was performed pre-GLP. A concise description of the protocol is available and results are reported clearly.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
no OECD scoring system was used
GLP compliance:
no
Remarks:
performed pre-GLP.
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 9 weeks
Vehicle:
unchanged (no vehicle)
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 ml
- Concentration: 100%
Duration of treatment / exposure:
Single dose / continuous exposure (no washing)
Observation period (in vivo):
Up to 7 days
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: Not performed

SCORING SYSTEM: Opacity, corneal swelling, chemosis, discharge, iritis, pannus and discomfort were checked in the treated animals on specific timepoints after instillation (up to recovery). No detailed information available. It is therefore not possible to derive scores from this study that can be directly related to the OECD scoring system. Expert judgment has to be used to evaluate the results with regard to the current classification criteria.
Irritation parameter:
cornea opacity score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
> 0 - < 1
Max. score:
4
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Remarks:
affecting 25-75% of corneal surface
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Remarks:
% swelling
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
> 10 - < 59
Max. score:
100
Reversibility:
fully reversible within: 7 days
Remarks on result:
positive indication of irritation
Remarks:
slight to moderate
Irritant / corrosive response data:
Litsea cubeba caused slight to moderate corneal lesions in two animals affecting between 25-75% of the corneal surface. These lesions were associated with slight to moderate corneal swelling. Opacity effects were also noted in two animals, but maximum grade was not >1. All animals developed slight to moderate conjunctivitis. All animals recovered within 7 days.

All three animals showed some irritating effects in the first days after exposure. Betwee day 2 and 7 after exposure recovery was completed in all animals. Reporting of the results was not in line with the current classification criteria and therefore have to be extrapolated based on expert judgement.

Interpretation of results:
other: Not irritating
Remarks:
based on the criteria outlined in 1272/2008/EC (CLP)
Conclusions:
Under the conditions of this study, Litsea cubeba oil was found to induce slight to moderate conjunctivitis in all test animals, but recovery was completed within 7 days. These results were extrapolated to the current classification criteria based on expert judgement. It was concluded that the test substance does not need to be classified for eye irritation based on the criteria outlined in 1272/2008/EC (CLP).
Executive summary:

An eye irritation study according to similar methods as described in OECD guideline 405 was performed with test substance Litsea cubeba essential oil. An amount of 0.1 ml of the undiluted test substance was instilled in one eye of three rabbits (no washing performed). Opacity, corneal swelling, chemosis, discharge, iritis, pannus and discomfort were checked in the treated animals on specific timepoints after instillation (15 minutes, 1 day, 2 days, 3 days and 7 days).

Litsea cubeba essential oil caused slight to moderate corneal lesions in two animals affecting between 25-75% of the corneal surface. These lesions were associated with slight to moderate corneal swelling. Opacity effects were also noted in two animals (not above 1). All animals developed slight to moderate conjunctivitis. All animals recovered within 7 days.

Under the conditions of this study, Litsea cubeba essential oil was found to induce slight to moderate conjunctivitis in all test animals, but recovery was completed within 7 days. These results were extrapolated to the current classification criteria based on expert judgement. It was concluded that the test substance does not need to be classified for eye irritation based on the criteria outlined in 1272/2008/EC (CLP).

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 June 1977 - 19 July 1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test was conducted according to methods similar to OECD guideline 405 and was performed pre-GLP. A concise description of the protocol is available and results are reported schematically.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
yes
Remarks:
only 3 animals were tested, no results on negative control reported
GLP compliance:
no
Remarks:
performed pre-GLP
Species:
rabbit
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks
Vehicle:
other: Tween 80
Controls:
no
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL
- Concentration: 50%
Duration of treatment / exposure:
Single dose / continuous exposure (no washing)
Observation period (in vivo):
Up to 21 days
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
No data

SCORING SYSTEM:
Opacity, corneal swelling, chemosis, discharge, iritis, pannus and discomfort were checked in the treated animals on specific timepoints after instillation (up to recovery). No detailed information is available. It is therefore not possible to derive scores from this study that can be directly related to the OECD scoring system. Expert judgment has to be used to evaluate the results with regard to the current classification criteria.
Irritation parameter:
cornea opacity score
Remarks:
average
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Score:
1.67
Max. score:
4
Reversibility:
not fully reversible within: 21 days
Irritation parameter:
iris score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Reversibility:
fully reversible within: 21 days
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Reversibility:
fully reversible within: 21 days
Remarks on result:
positive indication of irritation
Irritation parameter:
chemosis score
Basis:
animal: #1, #2, #3
Time point:
24/48/72 h
Reversibility:
fully reversible within: 21 days
Remarks on result:
positive indication of irritation
Irritant / corrosive response data:
Moderate corneal lesions affecting 25-100% of the corneal surface were observed in all animals. The lesions were associated with severe corneal swelling, moderate to severe conjunctivitis, slight to moderate discharge and iritis affecting 25-100% of the iris. Recovery was seen within 21 days. No results were reported for the negative control.

Summary of results:

Number of rabbits Discomfort Positive conjunctival reactions* Number with iritis Number with pannus Corneal opacity** Duration*** Maximum corneal swelling (%)****
Grade Opacity≥1 on day 8 Opacity≥0.5 on day 22
0.5 1 2 3 4 80-90 % 100%
3 - 3 3 0 - 1 2 - - 3 - 2 1
*Number of rabbits with conjuctival erythema or chemosis grades≥2
**Maximum grade obtained during period of observation
***Number of animals with corneal opacity grades≥1 on observation day 8 or with any ocular lesions on day 22 of the test
****Maximum swelling attained during period of observation

All three animals showed significant irritating effects in the first days after exposure. Between day 10 and 21 after exposure, recovery was completed in all animals. Reporting of the results was not in line with the current classification criteria and therefore data has to be extrapolated based on expert judgement.

Interpretation of results:
other: Irritating
Remarks:
based on the criteria outlined in 1272/2008/EC (CLP).
Conclusions:
Under the conditions of this study, Litsea cubeba oil was found to induce significant eye irritation in all test animals. Within 21 days all animals recovered. These results were extrapolated to the current classification criteria based on expert judgement. It was concluded that the test substance needs to be classified as an eye irritant (Cat. 2), based on the criteria outlined in 1272/2008/EC (CLP).
Executive summary:

An eye irritation study was performed according to a method similar to OECD 405 (Pre-GLP). Three 10 weeks old rabbits were exposed to a 50% litsea cubeba oil solution in Tween 80. Animals were observed 24 hours after treatment and at daily intervals therafter and graded for corneal, conjunctival and iridial damage.

Moderate corneal lesions affecting 25-100% of the corneal surface were caused in all animals. The lesions were associated with severe corneal swelling, moderate to severe conjunctivitis, slight to moderate discharge and iritis affecting 25-100% of the iris. Recovery was seen within 21 days in all animals.

Under the conditions of this study, Litsea cubeba oil was found to induce significant eye irritation in all test animals. These results were extrapolated to the current classification criteria based on expert judgment. It was concluded that the test substance needs to be classified as an eye irritant (Cat. 2), based on the criteria outlined in 1272/2008/EC (CLP).

Endpoint:
eye irritation: in vitro / ex vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation

Two studies were available for this endpoint and used in a Weight of Evidence approach, together with available information on the classification of the constituents of Litsea cubeba.

The first study was an acute dermal irritation study (method similar to OECD 404), in which Litsea Cubeba oil was applied semi-occlusively on the dorsal skin of four rabbits for 4 hours. The average scores for erythema at 24, 48 and 72 hour after dosing were 2.0 in all four animals whereas the average scores for edema at 24, 48 and 72 hour after dosing were 1.2, 1.3, 2.3 and 2.5.

The second study was also performed in accordance with a method similar to OECD 404 (performed pre-GLP). Major deviation was that no OECD scoring system was used to evaluate the erythema and oedema formation. Ten rabbits were exposed for 4 hours to the test substance (semi-occlusive) and skin reactions were qualitatively evaluated for 72 hours. Oedema was quite distinct to well-developed at 4 hours after exposure and slight to becoming well-developed at 24 hours. At 72 hours slight to becoming well-developed oedema was observed. Erythema was not scored at 4 and 24 hours, as the test sample stained the skin yellow. At 48 hours fairly distinct to well-developed erythema was noted, while it was classified as quite distinct to becoming well-developed at 72 hours. The qualitative results for oedema were translated to the OECD scoring system by the evaluator to be able to test them against the current classification criteria. This translation showed that 9 out of 10 animals had an oedema score of ≥2.3. It was not possible to derive an erythema score due to the lack of observations at one of the required timepoints.

The results of the first study show that in accordance with the new CLP criteria classification is not necessary, while the second study indicates that classification is required. Based on the fact that the main constituents of Litsea cubeba, Geranial, Neral and Limonene are also classified for skin irritation and represent typically 81% of the composition, classification of Litsea cubeba is considered required as well.

Eye irritation

Two studies were available for this endpoint and used in a Weight of Evidence approach, together with available information on the classification of the constituents of Litsea cubeba.

The first available eye irritation study was performed according to a method similar to OECD 405 (Pre-GLP). Three 10 weeks old rabbits were exposed to a 50% litsea cubeba oil solution in Tween 80. Animals were observed 24 hours after treatment and at daily intervals therafter and graded for corneal, conjunctival and iridial damage. Moderate corneal lesions affecting 25-100% of the corneal surface were caused in all animals. The lesions were associated with severe corneal swelling, moderate to severe conjunctivitis, slight to moderate discharge and iritis affecting 25-100% of the iris. Recovery was seen within 21 days in all animals. Under the conditions of this study, Litsea cubeba oil was found to induce significant eye irritation in all test animals.

In the second available eye irritation study according to a method similar as described in OECD guideline 405, an amount of 0.1 ml of the undiluted test substance was instilled in one eye of three rabbits (no washing performed). Litsea cubeba caused slight to moderate corneal lesions in two animals affecting between 25-75% of the corneal surface. These lesions were associated with slight to moderate corneal swelling. Opacity effects were also noted in two animals (not above 1). All animals developed slight to moderate conjunctivitis. All animals recovered within 7 days.

The results of both studies were extrapolated to the current classification criteria based on expert judgement, as no detailed information was available on the scoring system. As a result, in the first study Litsea cubeba was found to be irritating, while in the second study a negative result was found. Based on the fact that the main constituents of Litsea cubeba, Geranial and Neral are also classified for eye irritation and represent typically 69% of the composition, classification of Litsea cubeba is considered required as well.

Justification for classification or non-classification

Based on a Weight of Evidence approach and the classification of its main constituents, Litsea cubeba was concluded to have irritating properties in contact with skin. Therefore, the substances should be classified for skin irritation (Skin Irrit. 2 / H315) in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS).

Based on a Weight of Evidence approach and the classification of its main constituents, Litsea cubeba was concluded to have irritating properties in contact with eyes. Therefore, the substances should be classified for eye irritation (Eye Irrit. 2 / H319) in accordance with the criteria outlined in Annex I of 1272/2008/EC (CLP/EU-GHS).