Reaction Mass of 1,4-dimethyl-7-(prop-1-en-2-yl)-1,2,3,4,5,6,7,8-octahydroazulene and 3,8-dimethyl-5-(prop-1-en-2-yl)-1,2,3,3a,4,5,6,7-octahydroazulene and 4,8a,9,9-tetramethyldecahydro-1,6-methanonaphthalen-1-ol

Endpoint summary

Description of key information

Skin sensitizer (LLNA, 2008 and OECD 442D Keratinosens, 2020)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-03 to 2008-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

LLNA using lymph node cell count instead of [3H]-thymidine incorporation into lymph node cells.

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

not specified

Details on test animals and environmental conditions:

No data

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10, 25, 50, 100 % test material

No. of animals per dose:

Four

Details on study design:

- Four groups of four animals were treated for three consecutive days (days 1-3) via topical application to the dorsal surface of each ear.
- Test material was applied as 50 μL (25 μL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil (4:1).
- A group of four animals received the vehicle alone.
- A further group of four animals was treated with the positive control diluted at 25 % in the vehicle.
- On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting.

Positive control substance(s):

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

Statistics:

- The Stimulation Index (SI) was calculated from the formula SI = cell count of treated group / cell count of control group.

Positive control results:

- A Stimulation Index (SI) of 2.22 was recorded for the positive control diluted at 25 % in the vehicle.
- The SI result was more than 1.4 as expected.

Parameter:

SI

Value:

1.01

Test group / Remarks:

10%

Parameter:

SI

Value:

1.45

Test group / Remarks:

25 %

Parameter:

SI

Value:

2.24

Test group / Remarks:

50%

Parameter:

SI

Value:

2.53

Test group / Remarks:

100%

Cellular proliferation data / Observations:

The Stimulation Index (SI) calculated by pooled approach was respectively 1.01, 1.45, 2.24, and 2.53 for the treated groups at 10%, 25%, 50%, and 100%.

- The EC1.4 value determined by linear interpolation of points on the dose-response curve was 23.30 % (see document describing validation of EC value, attached).

- No mortality and no sign of systemic toxicity were noted in the test and control animals during the test.

- A moderate (+15.2%) to important (+41.6%) increase in ear thickness was recorded respectively at the concentration of 50% and 100%. An important increase in ear weight (+60.3%) was recorded at the concentration of 100%.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test material was considered to be a sensitiser under the conditions of the test and showed an EC1.4 of 23.30%.

Executive summary:

A study was performed in accordance with GLP and OECD Guideline 429 to assess the skin sensitisation potential of Patchouli Essential Oil in the CBA/J strain mouse following topical applications of the test material to the dorsal surface of the ear (Robertet Grasse, 2008). Four groups, each of four animals, were treated for three consecutive days with 50 μL (25 μL per ear) of the undiluted test item and the test item as a solution in Acetone/ Olive oil (4:1) at concentrations of 10%, 25%, 50% and 100% (v/v). The Stimulation Index, when using the pooled approach, is expressed as the cell count for each treatment group divided by the cell count of the control group and was 1.01, 1.45, 2.24 and 2.53 at concentrations of 10%, 25%, 50% and 100%, respectively.

The proliferation response of lymph node cells is expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item is regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values.

The Stimulation Index (SI) calculated by pooled approach was respectively 1.01, 1.45, 2.24,and 2.53 for the treated groups at 10%, 25%, 50%, and 100%.

The EC1.4 value determined by linear interpolation of points on the dose-response curve was 23.30%.

In view of these results, under these experimental conditions, the test item PATCHOULI EO was considered to be a sensitiser under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Key LLNA study (OECD 429 and GLP) :

A study was performed in accordance with GLP and OECD Guideline 429 to assess the skin sensitisation potential of Patchouli Essential Oil in the CBA/J strain mouse following topical applications of the test material to the dorsal surface of the ear (Robertet Grasse, 2008). Four groups, each of four animals, were treated for three consecutive days with 50 μL (25 μL per ear) of the undiluted test item and the test item as a solution in Acetone/ Olive oil (4:1) at concentrations of 10%, 25%, 50% and 100% (v/v). The Stimulation Index, when using the pooled approach, is expressed as the cell count for each treatment group divided by the cell count of the control group and was 1.01, 1.45, 2.24 and 2.53 at concentrations of 10%, 25%, 50% and 100%, respectively.

The proliferation response of lymph node cells is expressed as the number of lymphocytes per lymph node and as the ratio of lymphocytes into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item is regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values.

The Stimulation Index (SI) calculated by pooled approach was respectively 1.01, 1.45, 2.24,and 2.53 for the treated groups at 10%, 25%, 50%, and 100%. The EC1.4 value determined by linear interpolation of points on the dose-response curve was 23.30%.

In view of these results, under these experimental conditions, the test item PATCHOULI EO was considered to be a sensitiser under the conditions of the test.

Supportive in vitro Keratinosens (OCED 442D):

Three different PATCHOULI ESSENTIAL OIL qualities were tested in this study to investigate whether Patchouli oil is positive in KeratinoSens, and whether the result is dependent on different commercial grades. The oils were dissolved in cell culture medium and tested according to the standard operating procedure of the KeratinoSensTM assay at 12 concentrations in three repetitions, each time in three replicates. After a 48 h incubation time, luciferase induction and cellular viability at each of the concentrations were determined.

All three PATCHOULI ESSENTIAL OIL qualities were moderately toxic to the KeratinoSens™ cells. In all three repetitions, all three qualities did induce the luciferase gene above a threshold of 1.5. They are thus considered sensitizing according to the prediction model of the KeratinoSens™ assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data available and key results described in this summary, the substance has shown some skin sensitization potential and should therefore be classified as Skin Sensitizer Category 1B according to the (EC) No 1272/2008 Regulation (CLP).