Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-01-2006 to 19-05-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD 429 and EPA OPPTS 870.2600 and according to GLP guidelines.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

EC Number:

946-253-9

Molecular formula:

Not applicable

IUPAC Name:

Essential oil of Spearmint obtained from the aerial part of Mentha spicata and/or Mentha cardiaca (Lamiaceae) obtained by distillation

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Spearmint Oil
- Physical state: Liquid
- Lot/batch No.: Confidential information
- Expiration date of the lot/batch: Confidential information
- Storage condition of test material: Ambient temperature (under inert gas)

Specific details on test material used for the study:

TSCA-CAS No.: 8008-79-5

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Interfauna UK Limited, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 Weeks
- Weight at study initiation: no information available
- Housing: A maximum of 4 mice per cages. Environmental enrichment provided included tents, bases and nestlets.
- Diet (e.g. ad libitum): Ad libitum (RM1, supplied by Special Diet Services Limited, Witham, Essex, UK)
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days

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

IN-LIFE DATES: From: 18 January 2006 To: 24 January 2006

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 animals per group

Details on study design:

RANGE FINDING TESTS:
No information available

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA) in Mice (OECD 429)
- Criteria used to consider a positive response: A response was considered as positive if one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: No information available

Positive control substance(s):

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

Statistics:

The EC3 value was derived by interpolating between two points on the SI axis, one immediately above and the other immediately below the SI value of 3. The quantity applied per square centimetre was derived from this value, assuming that the area of the mouse ear is 1 cm2 and that 1 µL is 1 mg.

EC3 = [(3-d)/(b-d)] x (a-c) + c

EC3 = The estimated concentration of the test substance required to produce a 3-fold increase in the draining lymph node cell proliferative activity.
a = The concentration giving the SI immediately above 3
b = The SI of a
c = The concentration giving the SI immediately below 3
d = The SI of c
SI = Stimulation Index

Results and discussion

Positive control results:

For the concentrations of 10 and 15% w/v the response resulted in a greater than 3-fold increase in isotop incorporation with SI of 4.2 and 15.1 respectively.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.2

Test group / Remarks:

0.5%

Results 2

Parameter:

SI

Value:

1.1

Test group / Remarks:

1%

Results 3

Parameter:

SI

Value:

1.2

Test group / Remarks:

2.5%

Results 4

Parameter:

SI

Value:

1.9

Test group / Remarks:

5%

Results 5

Parameter:

SI

Value:

3.6

Test group / Remarks:

10%

Results 6

Key result

Parameter:

EC3

Value:

8.2

Remarks on result:

other: The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was 8.2% w/v (2050 µg/cm2).

Cellular proliferation data / Observations:

disintegrations per minute (DPM):
Vehicle only: 4635 0.5 % w/v Spearmint Oil: 5363 1% w/v Spearmint Oil: 5106 2.5% w/v Spearmint Oil: 5727 5% w/v Spearmint Oil: 8789 10% w/v Spearmint Oil: 16783

Any other information on results incl. tables

EC3 was calculated to be 8.2% (2050 µg/cm2).

Applicant's summary and conclusion

Interpretation of results:

other: Sensitising

Remarks:

Based on CLP criteria

Conclusions:

Under the test conditions, Spearmint Oil is classified as sensitising to skin, according to the criteria of Annex I of Regulation 1272/2008/EC (CLP).

Executive summary:

In a local lymph node assay performed in CBA/Ca strain mice according to OECD guideline 429 and in compliance with GLP, groups of mice (4 females/dose) were applied with 25 µL of Spearmint Oil at concentrations of 0 (vehicle control), 0.5, 1, 2.5, 5 or 10% w/v in ethanol/diethylphthalate (3:1 w/v) to the dorsal surface of each ear for three consecutive days. On Day 6, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and measured for radioactivity expressed as number of disintegrations per minute (DPM). The positive control substance hexylcinnamaldehyde (5, 10 and 25 % w/v) in acetone/olive oil (4:1 v/v) was tested in a similar was and used for positive control group.

 

Mean DPM for 0, 0.5, 1, 2.5, 5 and 10% Spearmint Oil were observed to be 4635, 5363, 5106, 5727, 8789 and 16783 dpm, respectively. Stimulation index for 0.5, 1, 2.5, 5 and 10% Spearmint Oil were calculated to be 1.2, 1.1, 1.2, 1.9 and 3.6 respectively. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was 8.2% w/v (2050 µg/cm2).

 

Under the test conditions, Spearmint Oil is classified as 'sensitising to skin', according to the criteria of Annex VI to Annex I of Regulation 1272/2008/EC (CLP).