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Description of key information

Skin sensitisation (GPMT): Sensitising (based on read across from its major constituent eugenol)

Skin sensitisation (LLNA): Sensitising (based on read across from its major constituent eugenol)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

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Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This publication describes a Guinea Pig Maximisation Test, performed according to methods resembling OECD guideline 406 (ref: Magnusson and Kligman, 1970). It was not indicated if GLP conditions were met in the original study. The study was assigned a Klimisch 2 rating due to the read-across purpose, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
Justification for type of information:
The read-across justification document is attached as a separate docuemt to the "Toxicological Information" summary of this dossier.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
not specified
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Existing study
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac, Oxon, UK
- Weight at study initiation: approx. 350 g
Route:
intradermal and epicutaneous
Vehicle:
other: dobs/saline (intradermal) and acetone/PEG (epicutaneous)
Concentration / amount:
Intradermal: 0.1%
Epicutaneous induction: 100%
Epicutaneous challenge: 25%
Route:
epicutaneous, occlusive
Vehicle:
other: dobs/saline (intradermal) and acetone/PEG (epicutaneous)
Concentration / amount:
Intradermal: 0.1%
Epicutaneous induction: 100%
Epicutaneous challenge: 25%
No. of animals per dose:
10
Details on study design:
RANGE FINDING TESTS:
Preliminary irritation tests were performed to determine suitable assay concentrations.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: 48 hours (epicutaneous)
- Test groups (n=10): Test article, six intradermal injections and one time epicutaneous application (6-8 days later)
- Control group (n=5): Vehicle only, six intradermal injections and one time epicutaneous application (6-8 days later)
- Site: Shoulder region

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: approx. 14 days after induction
- Exposure period: 24 hours
- Test/control group: Test article epicutaneously at maximum non-irritant concentration
- Evaluation (hr after challenge): 24 hours

C. SCORING SYSTEM
Scoring for erythema on a 0-3 scale
Challenge controls:
Not performed
Positive control substance(s):
no
Positive control results:
No positive control was included in the study.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle and 25% test article
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 and 25 % test article
No. with + reactions:
3
Total no. in group:
10

A sensitisation rate of 30% was calculated for the guinea pigs exposed to 25% Eugenol in the challenge phase.

Interpretation of results:
other: Sensitising
Remarks:
Based on CLP criteria
Conclusions:
Under the conditions of this study, positive skin reactions were noted in 3 out of 10 guinea pigs treated with 25% Eugenol at the challenge phase. A sensitisation rate of 30% was calculated. Based on the threshold value of 30% as mentioned in Annex I of the CLP Regulation (1272/2008/EC), the substance has to be classified as sensitising.
Executive summary:

This Guinea Pig Maximisation Test (GPMT) was performed to determine the sensitising potential of Eugenol. In the test group (n=10), intradermal induction was performed with 0.1% test article, epicutaneous induction with 100% test article and the final challenge exposure was done with 25% test article. A negative control group (n=5) was also included. Positive skin reactions were scored and sensitisation rates were calculated.

Positive skin reactions were noted in 3 out of 10 guinea pigs treated with 25% Eugenol at the challenge phase (mean score of 0.8). A corresponding sensitisation rate of 30% was calculated. No positive skin reactions were noted in the negative control animals.

Based on the results of this study and considering the threshold value of 30% as mentioned in Annex I of the CLP Regulation (1272/2008/EC), the substance has to be classified as sensitising.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This publication describes a Local Lymph Node Assay, performed according to methods resembling OECD guideline 429 (ref: Kimber and Basketter, 1992). It was not indicated if GLP conditions were met in the original study and there was no positive control included. The study was assigned a Klimisch 2 rating due to the read-across purpose, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.
Justification for type of information:
The read-across justification document is attached as a separate docuemt to the "Toxicological Information" summary of this dossier.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac, Oxon, UK
- Age at study initiation: 6-12 weeks
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0%, 5%, 10% and 25%
No. of animals per dose:
4
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay (LLNA) - pooled approach
- Criteria used to consider a positive response: SI ≥3 (according to OECD guideline 429)

TREATMENT PREPARATION AND ADMINISTRATION:
25 ul of 0%, 5%, 10% or 25% test article in vehicle applied to dorsum of ears, for three consecutive days.
After five days mice were injected IV with 20 uCi of 3-HTdR in 250 ul PBS.
Positive control substance(s):
other: not included in this test
Statistics:
Not performed
Positive control results:
Not included
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Derived from graph (estimated pooled dpm/node per dose group): - 0%: 0.05 - 5%: 0.06 - 10%: 0.12 - 25%: 0.4
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
8
Test group / Remarks:
25%
Interpretation of results:
other: Sensitising
Remarks:
Based on CLP criteria
Conclusions:
Under the conditions of this study, mice exhibited a dose dependent induction of proliferation in the lymph nodes as a result of exposure to Eugenol. A stimulation index (SI) of >3 was found only for the 25% dose group. It can be concluded that Eugenol is a sensitising substance at this concentration, based on the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC)
Executive summary:

This Local Lymph Node Assay (similar to OECD 429) was conducted to determine the sensitising potential of Eugenol in mice. Lymph node proliferation was determined after exposure to 0%, 5%, 10% or 25% Eugenol in vehicle, using beta-scintillation counting (dpm/node). A pooled approach (per test group) was used. Stimulation indices were calculated.

Measured disintegrations per node (pooled) were 0.05, 0.06, 0.12 and 0.4 for the 0%, 5%, 10% and 25% dosing groups, respectively. Corresponding stimulation indices (SI) were calculated to be 1.2, 2.4 and 8 for the 5%, 10% and 25% concentration, respectively.

Under the conditions of this study, mice exhibited a significant proliferation in the lymph nodes as a result of exposure to Eugenol. A stimulation index (SI) of >3 was found only for the 25% dose group. It can be concluded that Eugenol is a sensitising substance at this concentration, based on the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC)

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The read-across justification document is attached as a separate docuemt to this endpoint record
Reason / purpose for cross-reference:
read-across source
Justification for non-LLNA method:
Existing study
Positive control results:
No positive control was included in the study.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 and 25 % test article
No. with + reactions:
3
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle and 25% test article
No. with + reactions:
0
Total no. in group:
5

A sensitisation rate of 30% was calculated for the guinea pigs exposed to 25% Eugenol in the challenge phase.

Interpretation of results:
other: Sensitising
Remarks:
Based on CLP criteria
Conclusions:
A Guinea Pig Maximisation Test (GPMT) was performed to determine the sensitising potential of the source substance Eugenol, the results are read-across to the target substance Cinnamon Leaf oil. Under the conditions of this study, positive skin reactions were noted in 3 out of 10 guinea pigs treated with 25% Eugenol at the challenge phase. A sensitisation rate of 30% was calculated. Based on the threshold value of 30% as mentioned in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, the substance has to be classified as sensitising.
Executive summary:

A Guinea Pig Maximisation Test (GPMT) was performed to determine the sensitising potential of the source substance Eugenol, the results are read-across to the target substance Cinnamon Leaf oil. In the test group (n=10), intradermal induction was performed with 0.1% test article, epicutaneous induction with 100% test article and the final challenge exposure was done with 25% test article. A negative control group (n=5) was also included. Positive skin reactions were scored and sensitisation rates were calculated.

Positive skin reactions were noted in 3 out of 10 guinea pigs treated with 25% Eugenol at the challenge phase (mean score of 0.8). A corresponding sensitisation rate of 30% was calculated. No positive skin reactions were noted in the negative control animals.

Based on the results of this study and considering the threshold value of 30% as mentioned in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC, the substance has to be classified as sensitising.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The read-across justification document is attached as a separate docuemt to this endpoint record
Reason / purpose for cross-reference:
read-across source
Positive control results:
Not included
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Derived from graph (estimated pooled dpm/node per dose group): - 0%: 0.05 - 5%: 0.06 - 10%: 0.12 - 25%: 0.4
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
2.4
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
8
Test group / Remarks:
25%
Interpretation of results:
other: Sensitising
Remarks:
Based on CLP criteria
Conclusions:
A Local lymph node assay was performed to determine the sensitising potential of the source substance Eugenol, the results are read-across to the target substance Cinnamon Leaf oil. Under the conditions of this study, mice exhibited a dose dependent induction of proliferation in the lymph nodes as a result of exposure to Eugenol. A stimulation index (SI) of >3 was found only for the 25% dose group. It can be concluded that Eugenol is a sensitising substance at this concentration, based on the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC)
Executive summary:

This Local Lymph Node Assay (similar to OECD 429) was conducted to determine the sensitising potential of Eugenol in mice the results are read-across to the target substance Cinnamon Leaf oil. Lymph node proliferation was determined after exposure to 0%, 5%, 10% or 25% Eugenol in vehicle, using beta-scintillation counting (dpm/node). A pooled approach (per test group) was used. Stimulation indices were calculated.

Measured disintegrations per node (pooled) were 0.05, 0.06, 0.12 and 0.4 for the 0%, 5%, 10% and 25% dosing groups, respectively. Corresponding stimulation indices (SI) were calculated to be 1.2, 2.4 and 8 for the 5%, 10% and 25% concentration, respectively.

Under the conditions of this study, mice exhibited a significant proliferation in the lymph nodes as a result of exposure to Eugenol. A stimulation index (SI) of >3 was found only for the 25% dose group. It can be concluded that Eugenol is a sensitising substance at this concentration, based on the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC)

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

The sensitising potential of cinnamon leaf oil was based on read across to its major constituent eugenol. Both an LLNA and a guinea pig maximisation test are available for eugenol, which are both included in a weight-of-evidence approach.


 


In the Local Lymph Node Assay (similar to OECD 429), lymph node proliferation was determined after exposure to 0%, 5%, 10% or 25% eugenol in vehicle, using beta-scintillation counting (dpm/node). A pooled approach (per test group) was used.


Under the conditions of this study, mice exhibited a significant proliferation in the lymph nodes as a result of exposure to Eugenol. A stimulation index (SI) of >3 was found only for the 25% dose group. It can be concluded that Eugenol is a sensitising substance at this concentration, based on EU-criteria.


 


In the Guinea Pig Maximisation Test (GPMT), intradermal induction was performed with 0.1% test article, epicutaneous induction with 100% test article and the final challenge exposure was done with 25% test article. A negative control group (n=5) was also included. Positive skin reactions were scored and sensitisation rates were calculated. Positive skin reactions were noted in 3 out of 10 guinea pigs treated with 25% eugenol at the challenge phase (mean score of 0.8). A corresponding sensitisation rate of 30% was calculated. No positive skin reactions were noted in the negative control animals. Based on the results of this study and considering the threshold value of 30% as mentioned in EU-criteria, the substance has to be classified as sensitising.



Migrated from Short description of key information:
Mouse Local Lymphnode Assay (LLNA): sensitising (equivalent or similar to OECD429)
Guinea Pig Maximisation Test: sensitising (equivalent or similar to OECD406)

Justification for classification or non-classification

Based on the available results, cinnamon leaf oil needs to be classified as a skin sensitiser, in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).