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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 06, 1995 - July 18, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

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:
other: acetone
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)

Resultsopen allclose all
Parameter:
SI
Value:
1.3
Test group / Remarks:
1% w/v
Parameter:
SI
Value:
1.8
Test group / Remarks:
10% w/v
Parameter:
SI
Value:
0.91
Test group / Remarks:
30% w/v
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 test substgance (% w/v)

Number of lymph nodes assayed

Count per minute (cpm)

cpm/lymph nodes (x102)

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:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
The test substance is not considered to be a moderate or strong skin sentizer und the conditions of this test.
Executive summary:

The increase in isotope incorporation was less than 3-fold at all concentrations. Also the response was not consistent with a biological dose-response. Consequently, L-menthol has not fulfilled the criteria for a moderate or strong sensitiser under the conditions of the test.