p-mentha-1,3-diene

Endpoint summary

Description of key information

In the key in vivo study equivalent or similar to OECD TG 429, the murine LLNA ([3H]thymidine incorporation assay was performed to assess the sensitizing potency of alpha terpinene. Alpha terpinene was tested in five different concentrations of 1, 5, 10, 15 and 25 (% w/v) using mice in groups of three.

The results were expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI), i.e., test group/control group ratio. Test material that at one or more concentrations gave rise to a SI greater than 3 were considered to be sensitizers in the LLNA.

The results of this assay showed, that the SI was greater than 3 at the concentrations of 10, 15 and 25 % of alpha terpinene. Based on these results, alpha terpinene was shown to be a sensitizer of moderate potency with EC3 value of 0.65 M (8.9 % w/v).

The results of the supportive study of Rudback et al, 2012 have demonstrated that alpha terpinene was able to auto-oxidise rapidly on exposure to air, and thereby to acquire strong skin sensitising potential, with an EC3 of approximately 1%.   The study was shown, that the concentration of alpha terpinene decreased rapidly when exposed to air at room temperature. The concentration of alpha terpinene had decreased to 53% after 10 days and 21% after 24 days, and after 66 days, alpha terpinene was not detected in the oxidation mixture (method limit of detection 0.2%).

In this study, the murine LLNA equivalent or similar to OECD TG 429 was used to determine the sensitization potency of the air-exposed alpha terpinene.  

The following EC3 values were afforded for the air-exposed alpha terpinene: three weeks oxidized alpha terpinene: 0.9% w/v (content of pure a-terpinene: ca. 20%); seven weeks oxidized alpha terpinene: 1.0% w/v (content of pure alpha terpinene: ca. 2%).

Based upon the available data summarised above, the conclusion drawn is that alpha-terpinene does have the potential to cause skin sensitisation and allergic contact dermatitis, and that this potential is acquired, and more probably enhanced, by auto-oxidation on the skin, and/or enzymatic activation in the skin.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

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)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

Alpha-Terpinene (90%, tech.) was purchased from Acros Organics (Geel, Belgium).

The purity of purchased test compound was determined to be >=98% (GC/MS) before testing. Alpha terpinene was purified by column chromatography on silica gel (100% n-pentane and 5% diethyl ether in hexanes, respectively).

Species:

mouse

Strain:

not specified

Sex:

not specified

Details on test animals and environmental conditions:

not specified

Vehicle:

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

Concentration:

1, 5, 10, 15, 25 (% w/v)

No. of animals per dose:

3 animals per concentration

Details on study design:

PRE-SCREEN TESTS: not specified

MAIN STUDY - murine LLNA assay

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: [3H]thymidine incorporation (dpm/lymph node)
- Criteria used to consider a positive response: The sensitizing potency of the test compounds was classified according to the following: EC3 < 0.1%, extreme; EC3 0.1-<1%, strong; EC3; EC3 1-<10%, moderate; and EC3 10-<100%, weak (ECETOC (2003), Kimber et al. (2003)).

Positive control substance(s):

other: see remarks

Statistics:

Results are expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI), i.e., test group/control group ratio. Test material that at one or more concentrations caused the SI greater than 3 was considered to be positive in the LLNA. EC3 value (the estimated concentration required to induce an SI of 3) was calculated by linear interpolation.

Positive control results:

The results of this study were compared with the prohaptenic cinnamic alcohol and its reactive metabolite cinnamic aldehyde, which have EC3 values of 1.5 (20.1%) and 0.10 M (1.3%), respectively.

Key result

Parameter:

EC3

Value:

ca. 8.9

Remarks on result:

other: sensitizer of moderate potency

Key result

Parameter:

SI

Value:

ca. 3.4

Test group / Remarks:

3 animals

Remarks on result:

other: test concetration 10%

Key result

Parameter:

SI

Value:

ca. 8.9

Test group / Remarks:

3 animals

Remarks on result:

other: test concetration 15%

Key result

Parameter:

SI

Value:

ca. 23

Test group / Remarks:

3 animals

Remarks on result:

other: test concetration 25%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA - not specified

DETAILS ON STIMULATION INDEX CALCULATION : The SI corresponds to the increase in thymidine incorporation of treated groups relative to vehicle-treated control.

EC3 CALCULATION : EC3 values (the estimated concentration required to induce an SI of 3) were calculated using linear interpolation.

CLINICAL OBSERVATIONS: not specified

BODY WEIGHTS: not specified

Table 1. LLNA results for alpha terpinene
Alpha terpinene and test concentrations (% w/v)	[3H]thymidine incorporation (dpm/lymph node)	SI	EC3 value of   Alpha terpinene and test concentrations (% w/v)		sensitizing potency
			% w/v	M
0	755		8.9	0.65	moderate
1	848	1.1
5	1153	1.5
10	2595	3.4
15	6740	8.9
25	17176	23

Interpretation of results:

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

Conclusions:

Based on the results of this study, alpha terpinene was shown to be sensitizer of moderate potency with the EC3 value of 0.65 M (8.9 % w/v). The SI was greater than 3 at the concentrations of 10, 15 and 25 %.

Executive summary:

In this study, the murine LLNA ([3H]thymidine incorporation assay was used to assess the sensitizing potency of alpha terpinene. Alpha terpinene was tested in five different concentrations of 1, 5, 10, 15 and 25 (% w/v) using mice in groups of three.

The results were expressed as mean dpm/lymph node for each experimental group and as stimulation index (SI), i.e., test group/control group ratio. Test material that at one or more concentrations gave rise to an SI greater than 3 were considered to be sensitizers in the LLNA.

The results of this study were compared with the prohaptenic cinnamic alcohol and its reactive metabolite cinnamic aldehyde, which have EC3 values of 1.5 (20.1%) and 0.10 M (1.3%), respectively.

The results demonstrated, that the SI was greater than 3 at the concentrations of 10, 15 and 25 % of alpha terpinene. Based on these results, alpha terpinene was shown to be a sensitizer of moderate potency with EC3 value of 0.65 M (8.9 % w/v).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the in vivo studies equivalent or similar to OECD TG 429 (murine LLNA), alpha terpinene should be classified as a skin sensitizer category 1 according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.