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Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 29 to June 6, 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 429 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:

In vivo test system

Test animals

Details on test animals and environmental conditions:
-Source: Jackson Laboratories, Bar Harbor, ME 04609
-Age at study initiation: 8-9 weeks
-Weight at study initiation: 18-23 g
-Housing: 5/cage
-Diet (e.g. ad libitum): ad libitum
-Water (e.g. ad libitum): ad libitum
-Acclimation period: 6 days

-Temperature (ºC): 21.7-27.7
-Humidity (%): 28-68
-Air changes (per hour):
-Photoperiod (hrs dark/hrs light): 12/12

Study design: in vivo (LLNA)

other: 3:1 diethyl phthalate/ethanol
2.5%, 5.0%, 10%, 25% and 50% (v/v)
No. of animals per dose:
Details on study design:
The purpose of the study was to determine if the test material would induce a hypersensitivity response in mice as measured by the proliferation of lymphocytes in the draining lymph nodes. A total of 45 CBA/J female mice were divided into 9 groups consisting of 5 animals in each group. Groups included 1 control (vehicle) group (diethyl phthalate/ethanol in a ratio of 3:1 ratio); 3 positive control groups and 5 test groups (2.5%, 5.0%, 10%, 25% and 50% (v/v) OTNE with the vehicle). The control group was treated with the vehicle only. Animals were checked daily for mortality on Day 1 to 6, for clinical observations immediately prior to dose administration, immediately post-dose on Days 1-3 and then once daily on Days 4-6, and for erythema and oedema using the Draize scoring system. Animals were also weighed daily on Days 1-6. A volume of approximately 25 µl of 5 different concentrations of OTNE (%v/v) in a vehicle of 3:1 ethanol:diethyl phthalate was applied topically to the dorsum of each ear lobe (left and right) once per day for three consecutive days. There were 24±2 hours between applications of test material. Three days after the third topical application (Day 6) all mice were injected intravenously into a tail vein with 250 µl of sterile saline containing approximately 20 µCi [3]H-thymidine. Five hours after the intravenous injection, the animals were sacrificed. The draining auricular lymph nodes were removed. At removal, the number of nodes collected per animal was recorded and the nodes were examined for size/appearance and the data recorded. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2-8ºC. The pellets were recovered by centrifugation and resuspended in 1 ml of TCA and transferred to a vial containing scintillation fluid. An additional 1 ml of TCA was used to rinse the tube and it was also transferred to the scintillation fluid. Incorporation of the [3]H-thymidine was measured in a beta-scintillation counter. The mean DPM per node for each group was evaluated. Increases in [3]H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test material elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control. Individual DPM values were analyzed by log transformation (base 10) of the data. If the data indicated that the test material was positive, the EC3 (estimated concentration of the test material required to produce a 3-fold increase in the draining lymph node cell proliferative activity) was calculated by the following formula: EC3 = c+[(3-d)/(b-d)](a-c) where the data points lying immediately above and below the SI value of 3 have the coordinates (a,b) and c,d) respectively. Body weight data were also evaluated.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
The evaluation of the equality of means for body weight data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means were found, a Dunnett's test was used to determine the degree of significance from the control means.

Results and discussion

Positive control results:
Stimulation indices at 5, 15, and 35% were 5.2, 4.4, and 3.8, respectively. Mean DPM at 0 (vehicle), 5, 15, and 35% was 1694, 8892, 7372, and 6422, respectively.

In vivo (LLNA)

Resultsopen allclose all
Key result
Remarks on result:
other: Stimulation indices at 2.5, 5, 10, 25, and 50% were 1.4, 2.4, 5.2, 28.9, and 13.5, respectively. EC3 is calculated to be 6.07%
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM at 0 (vehicle), 2.5, 5, 10, 25, and 50% were 1694, 2412, 3994, 8726, 48992, and 22879, respectively.

Applicant's summary and conclusion

Interpretation of results:
other: Sensitising Category 1B
according to EU CLP (EC 1272/2008 and its amendments)
Under the conditions of this study, OTNE was considered to have skin sensitising activity with an EC3 of 6.07% and a NOEC of 2.5%.
Executive summary:

In a local lymph node assay according to OECD TG 429, groups of mice were topically treated on the dorsal surface of both ears with 0 (vehicle), 2.5, 5.0, 10, 25 or 50% (v/v) OTNE in 3:1 diethyl phthalate/ethanol daily for 3 days. On the 6thday, mice were injected intravenously with [3]H-thymidine and 5 hours later were euthanized. The draining auricular lymph nodes were extracted and prepared for determination of [3]H-thymidine content. All mice survived to termination. Stimulation indices at 2.5, 5, 10, 25, and 50% were 1.4, 2.4, 5.2, 28.9, and 13.5, respectively. OTNE was considered to have skin sensitising activity with an EC3 of 6.07%, indicating the concentration at which a stimulation index of 3 is observed. A NOEC of 2.5% was derived.