(+)-menthol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Study period:

April 06, 1995 - july 18, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail. Performed on industrial lot under OECD 429 guidelines and GLP compliance

Data source

Reference

Reference Type:

other company data

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

other: Kimber, I, Hilton, J., Weisenberger, C., The murine local lymph node assay for identification of contact allergens: A preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis, 21, 215- 220, 19

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

L-menthol

EC Number:

218-690-9

EC Name:

L-menthol

Cas Number:

2216-51-5

IUPAC Name:

2-isopropyl-5-methylcyclohexanol

Constituent 2

Reference substance name:

Cyclohexanol, 5-methyl-2-(1-methylethyl)-, [1R-(1.α.,2.β.,5.α.)]-

IUPAC Name:

Cyclohexanol, 5-methyl-2-(1-methylethyl)-, [1R-(1.α.,2.β.,5.α.)]-

Details on test material:

Name : HR 95/620009
Physical state : white crystalline solid
Storage condition and handling : cool, dry place

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS:
- Strain: CBA/Ca/01a/Hsd strain
- Sex and number: 4 males per dose
- Source: Harlan Olac Limited, Blackthorne, Bicester, Oxon, UK
- Age: young adults
- Weight at study initiation: no data
- Controls: yes, with acetone

ADMINISTRATION/EXPOSURE
- Concentrations used were: 0 (vehicle), 1 %, 10 %, 30 %
- Positive control: Positive control study with hexylcinnamaldehyde (3 and 10% in acetone)

ENVIRONMENTAL CONDITIONS :
- The animal room temperature was 21±2°C
- Relative humidity of 55±15%
- 12-hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Concentrations used were: 0 (vehicle), 1 %, 10 %, 30 %

No. of animals per dose:

4 mice in the treatment group and 4 mice served as the control

Details on study design:

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION :
In the local lymph node assay, groups of CBA/Ca mice (4/group) were topically treated on the dorsum of each ear with 25 µl of 0 (vehicle control), 1, 10, or 30% test substance in acetone daily for 3 consecutive days. Mice were housed up to 4 animals/cage by dose group and given feed and water ad libitum.

(3)H-methyl thymidine Administration :
Mice were intravenously (tail vein) injected with 250 µl of phosphate buffered saline containing 20 µCi of a 2.0 Ci/mmol specific activity (3)H-methyl thymidine 3 days after the last topical application.

TERMINATION :
Five hours later, animals were euthanized by halothane inhalation and cervical dislocation. Draining auricular lymph nodes were removed, pooled with nodes from the same dose group, and placed in phosphate buffered saline.

PREPARATION OF SINGLE CELL SUSPENSION :
Mechanical disaggregation through 200-mesh stainless steel gauze was employed to prepare a single cell suspension. Afterwards, the cell suspension was washed 3x by centrifugation and addition of phosphate buffered saline. Then, the supernatant was removed and 3 ml of 5% trichloroacetic acid was added. The suspension was left overnight at 4°C for precipitation and then centrifuged to produce sample pellets. The supernatant was removed and the pellet (cells) was resuspended using 1 ml trichloroacetic acid.

DETERMINATION OF THE (3)H-methyl thymidine INCORPORATION :
The samples underwent ß-scintillation counting using a Packard Tri-Carb 2000CA Liquid Scintillation Counter.

INTERPRETATION OF THE RESULTS
Results were expressed as mean counts per minute (cpm) value per lymph node. A test:control ratio was calculated by dividing the activity of each test group by that of the vehicle control group. If the results show (1) an increase of 3-fold or more in isotope incorporation for at least one concentration when compared with the vehicle control; and (2) a dose-response, then the test substance is designated as a potential sensitizer.

Positive control substance :
A positive control study with hexylcinnamaldehyde (CAS No 101-86-0) (3 and10% in acetone) was previously conducted in a similar manner to verify the capacity of the strain of mouse to respond to a known sensitizing chemical. The positive control was concluded to be a potential sensitizer in this assay.

Positive control substance(s):

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

Results and discussion

Positive control results:

1 % ( w/v) : cpm/lymph node= 2.34x10(2) Result : negative
10 % ( w/v) : cpm/lymph node= 6.65x10(2) Result : positive
30% ( w/v) : cpm/lymph node= 8.39x10(2) Result : positive
                 

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: 1 % w/w : cpm/lymph node=2.02x10(2) Result : negative 10 % ( w/v) : cpm/lymph node= 2.79x10(2) Result : negative 30% ( w/v) : cpm/lymph node= 1.41x10(2) Result : negative

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 1 % w/v : Counts per minute (cpm)=1212 10 % no effects : Counts per minute (cpm)=2234 30 % no effects : Counts per minute (cpm)=1131

Any other information on results incl. tables

MAIN TEST :

Concentration of HR 95/620009 (% w/v)	Number of lymph nodes assayed	Count per minute (cpm)	cpm/lymph nodes (x10^2)	test : control ration
0 (vehicle)	8	1236	1.55	N/A
1	6	1212	2.02	1.30
10	8	2234	2.79	1.80
30	8	1131	1.41	0.91

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

HR 95/620009 should be considered unlikely to be a moderate or strong skin sentizer