Essential oil of Ylang Ylang III obtained from the flowers of Cananga odorata (Annonaceae) by steam distillation

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August 2006 - 5 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The justification document for read-across is attached to this record.

05 February 2018 READ-ACROSS STUDY / YLANG YLANG III OIL - SKIN SENSITISATION I&B9W8768R001F0.2

According to Annex VII, 8.3 of the REACh Regulation (EC) No 1907/2006, skin sensitisation is standard information required for the registration of substances manufactured or imported in quantities of one tonne per year or more. However, according to Annex XI, 1.5 of the REACH Regulation, Read-across and grouping approaches can be used to adapt the standard testing regime. This read-across study report follows notably the recommendations made by the European Chemicals Agency in its “Guidance on information requirements and chemical safety assessment Chapter R.6 – QSARs and grouping of chemicals” (ECHA, 2008) and in its document “Read-Across Assessment Framework (RAAF)” (ECHA, 2017).

A read-across approach appears appropriate to predict the endpoint “skin sensitisation” for the substance Ylang Ylang III oil because:

An LLNA study, according to OECD test guideline 429, is available for the substance Ylang Ylang Ext, which composition is very similar to the target substance.
Ylang Ylang Ext /I /II oil and Ylang Ylang III oil, are very similar in composition and show mainly variations in concentrations of constituents. A large number of these constituents is notified for skin sensitisation, and the QSAR toolbox gives various alerts for reactive structures among the constituents which could promote initiation of the adverse outcome pathway for skin sensitisation.
The source substance Ylang Ylang Ext /I /II oil has been classified for skin sensitisation, and based on the composition similarity regarding reactive constituents of the source and target substance, the results for skin sensitisation can be read across to the target substance.

This report follows the RAAF method and so presents:
1) The hypothesis: analogue read-across approach, based on the similarity of the structures and the skin sensitising potential for these types of structures;
2) The scientific justifications (“Assessment Elements”) and their evaluation (“Assessment Options”); which demonstrate the confidence that can be put in this prediction.
3) The conclusions, usable for classification assessment or risk assessment, which are summarised hereafter.

Reason / purpose for cross-reference:

read-across source

Positive control results:

The stimulation index at tested concentrations 5, 10 and 25% w/v were 1.7, 2.3, 6.4, respectively. This result confirms the validity of the test.

Key result

Parameter:

EC3

Remarks:

%

Value:

6.8

Parameter:

SI

Value:

1.5

Test group / Remarks:

0.5% concentration

Parameter:

SI

Value:

1.4

Test group / Remarks:

1% concentration

Parameter:

SI

Value:

2.1

Test group / Remarks:

2.5% concentration

Parameter:

SI

Value:

2.5

Test group / Remarks:

5% concentration

Parameter:

SI

Value:

3.9

Test group / Remarks:

10% concentration

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Ylang Ylang Extra EO Comores induced a higher than 3-fold increase in lympocyte proliferation at the highest concentration tested (10% w/v).

DETAILS ON STIMULATION INDEX CALCULATION
Mean disintegrations per minute (DPM) values were:
- Vehicle control: 533
- 0.5%: 805
- 1%: 742
- 2.5%: 1099
- 5%: 1331
- 10%: 2090

EC3 CALCULATION
The calculated EC3 was 6.8%.

CLINICAL OBSERVATIONS:
No visual levels of skin irritancy were observed at any of the concentrations used on or around the esar area for the duration of the study.

BODY WEIGHTS
Body weights were recorded but showed no abnormalities.

Interpretation of results:

other: Skin Sensitiser 1B

Remarks:

Based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Ylang Ylang Extra EO Comores induced a higher than 3-fold increase in lympocyte proliferation at the highest concentration tested (10% w/v). The calculated EC3 was 6.8%. This result is used for Ylang Ylang III oil, which needs to be classified as Skin Sensitiser Category 1B in accordance with Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

A Local Lymph Node Assay (LLNA) was performed according to the OECD guideline 429 (2002). Four mice per group were treated with 0%, 0.5%, 1%, 2.5%, 5%, and 10% w/v Ylang Ylang Extra EO Comores in 1:3 ethanol:diethyl phthalate (vehicle) for three consecutive days, followed by in vivo radiolabelling (by injection) and lymphocyte proliferation analysis with the liquid scintillation counter. The number of disintegrations per minute (dpm) was determined for each group for the pooled lymph nodes. Treatment with Ylang ylang extra EO Comores did induce significantly the lymph node proliferation in mice as compared to controls at the highest dose tested. A higher than 3-fold increase in lympocyte proliferation was found at the highest concentration tested (10% w/v). The calculated EC3 was 6.8%. This result is used for Ylang Ylang III oil, which needs to be classified as Skin Sensitiser Category 1B in accordance with Annex I of the CLP Regulation (1272/2008/EC).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2002

Deviations:

no

Remarks:

Remarks: No dose selection rationale is provided. Information on solubility of the substance in the solvent is not given.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Ylang-ylang, ext.

EC Number:

281-092-1

EC Name:

Ylang-ylang, ext.

Cas Number:

83863-30-3

IUPAC Name:

Essential oil of Ylang Ylang Ext/I/II obtained from the flowers of Cananga odorata (Annonaceae) by steam distillation

Test material form:

other: liquid

Remarks:

yellow

Details on test material:

- Name of test material (as cited in study report): Ylang Ylang extra EO Comores
- Physical state: liquid
-Colour: yellow
- Analytical purity: not specified (pure material)
- Storage condition of test material: ambient temperature in the dark

Specific details on test material used for the study:

- Name of test material (as cited in study report): Ylang Ylang extra EO Comores
- CTL test substance reference No: Y13877/001
- Expiration date of the lot/batch: June 2007
- Physical state: liquid
- Colour: yellow
- Analytical purity: not specified (pure material)
- Storage condition of test material: ambient temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/Ca/Ola/Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan UK Limited, Shaw's Farm, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8-12 weeks
- Housing: 4 mice/cage, suitable cages
- Diet: ad libitum, RM1 by Special Diet Services Limited, Witham, Essex, UK
- Water: ad libitum
- Acclimation period: 5 days prior dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): min 15
- Photoperiod (hrs dark / hrs light): 12

Study design: in vivo (LLNA)

Vehicle:

other: 1:3 ethanol:diethylphthalate

Concentration:

0.5, 1, 2.5, 5 and 10% (w/v)

No. of animals per dose:

4

Details on study design:

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the substance was applied on the dorsal ear with a micro-pipette (injection), once for three consecutive days. Dose preparations were used within 24 hours. Three days after last treatment, 250 µl PBS 20 µCi/mmol specific activity 3H-methyl thymidine, were injected in the tail vein. Five hours thereafter the animals were sacrificed.

Cell suspension: mechanical disaggregation of pooled lymph nodes; properly prepared as described in the Guideline OECD 429.
Incorporation of 3H-methyl thymidine was measured by β-scintillation (disintegrations/min: DPM).

Criteria used to consider a positive response:
- Stimulation Index (SI) was calculated as Disintegrations per min (DPM) per concentration/DPM vehicle control. A positive response is indiated when the SI ≥ 3.
- The EC3 is the concentration for which a 3- fold increase in DPM is seen. The EC3 is calculated as follows: [(3-d)/(b-d)]* (a-c)+c, where a: concentration giving the SI immediately higher than 3, b: SI of a, c: concentration giving an SI immediately below 3, d: SI of c

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

The stimulation index at tested concentrations 5, 10 and 25% w/v were 1.7, 2.3, 6.4, respectively. This result confirms the validity of the test.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Remarks:

%

Value:

6.8

Results 2

Parameter:

SI

Value:

1.5

Test group / Remarks:

0.5% concentration

Results 3

Parameter:

SI

Value:

1.4

Test group / Remarks:

1% concentration

Results 4

Parameter:

SI

Value:

2.1

Test group / Remarks:

2.5% concentration

Results 5

Parameter:

SI

Value:

2.5

Test group / Remarks:

5% concentration

Results 6

Parameter:

SI

Value:

3.9

Test group / Remarks:

10% concentration

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Ylang Ylang Extra EO comores induced a higher than 3-fold increase in lympocyte proliferation at the highest concentration tested (10% w/v).

DETAILS ON STIMULATION INDEX CALCULATION
Mean disintegrations per minute (DPM) values were:
- Vehicle control: 533
- 0.5%: 805
- 1%: 742
- 2.5%: 1099
- 5%: 1331
- 10%: 2090

EC3 CALCULATION
The calculated EC3 was 6.8%.

CLINICAL OBSERVATIONS:
No visual levels of skin irritancy were observed at any of the concentrations used on or around the esar area for the duration of the study.

BODY WEIGHTS
Body weights were recorded but showed no abnormalities.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sensitiser 1B

Remarks:

Based on CLP criteria (Annex I of 1272/2008/EC)

Conclusions:

Ylang Ylang Extra EO comores induced a higher than 3-fold increase in lympocyte proliferation at the highest concentration tested (10% w/v). The calculated EC3 was 6.8%. Under the conditions of this assay, the test substance is needs to be classified as Skin Sensitiser Category 1B in accordance with Annex I of the CLP Regulation (1272/2008/EC).

Executive summary:

A Local Lymph Node Assay (LLNA) was performed according to the OECD guideline 429 (2002). Four mice per group were treated with 0%, 0.5%, 1%, 2.5%, 5%, and 10% w/v Ylang Ylang Extra EO Comores in 1:3 ethanol:diethyl phthalate (vehicle) for three consecutive days, followed by in vivo radiolabelling (by injection) and lymphocyte proliferation analysis with the liquid scintillation counter. The number of disintegrations per minute (dpm) was determined for each group for the pooled lymph nodes. Treatment with Ylang Ylang Extra EO Comores did induce significantly the lymph node proliferation in mice as compared to controls at the highest dose tested. A higher than 3-fold increase in lympocyte proliferation was found at the highest concentration tested (10% w/v). The calculated EC3 was 6.8%. Under the conditions of this assay, the test substance is needs to be classified as Skin Sensitiser Category 1B in accordance with Annex I of the CLP Regulation (1272/2008/EC).