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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Specific details on test material used for the study:
Identification: 2,3-epoxypropylo-tolylether
Batch: 30380-132
Purity: Approx. 90%
Physical state, appearance: Colourless liquid
Expiry Date: 12 November 2021
Storage Conditions: Room temperature

In vivo test system

Test animals

other: mouse
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
Number of animals for the pre-tests: 6 females (2 for each pre-test)
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): 8 - 13 weeks
Identification: The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by tail tags. In the pre-experiment, animals were identified by cage number.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
0.5%, 1%, and 2.5%
No. of animals per dose:
Details on study design:
Experimental Design and Study Conduct
Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.5, 1, and 2.5% in acetone/olive oil (4+1 v/v). The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm)of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20 μCi of 3H-methyl thymidine (equivalent to 79.9 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Preparation of Single Cell Suspensions
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.

Determination of ear weights
In the pre-test, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually.

Determination of Body Weights
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

Data Evaluation
Interpretation of raw data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

General Calculations
Where appropriate, the EC3 value were calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Positive Control Data
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1 v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2018.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

In vivo (LLNA)

Key result
Cellular proliferation data / Observations:
No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period. From days 2 and 3 the animals showed an erythema of the ear skin (Score 1).

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
The test item 2,3-epoxypropylo-tolylether was found to be a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item 2,3-epoxypropylo-tolylether formulated in acetone/olive oil (4+1 v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 0.5, 1, and 2.5%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by three pre-experiments.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From days 2 and 3 the animals showed an erythema of the ear skin (Score 1).

In this study Stimulation Indices (S.I.) of 1.58, 2.09, and 6.34 were determined with the test item at concentrations of 0.5, 1, and 2.5% (w/w) in acetone/olive oil (4+1 v/v), respectively. A clear dose response was observed.

The test item 2,3-epoxypropylo-tolylether was found to be a skin sensitiser and an EC3 value of 1.3 % was derived.