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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:
20 October 2004 to 7 December 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, B.V. Postbus 6174, NL-5960 AD Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16-24 g
- Housing: Individually in Makrolon type-2 cages
- Diet: Pelleted standard Kliba 3433 mouse maintenance diet, ad libitum
- Water: Community tap water, ad libitum
- Acclimation period: 6 days under test conditions after health examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: From: 20-Oct-2004 To: 01-Nov-2004

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: no data
- Irritation: Non-GLP pre-test in two mice, tested at 10, 25, 50 and 100% with acetone:olive oil (4:1) as the vehicle. 24 Hours after a single topical application, 100% (undiluted) was the highest technically applicable concentration while avoiding systemic toxicity and excessive local irritation in the chosen vehicle
- Lymph node proliferation response: not determined

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: local lymph node assay
- Criteria used to consider a positive response: 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) and 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.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µl of propenoic acid, tris(1-methylethyl)silyl ester was applied topically (epidermally) to the entire dorsal surface of each ear lobe (left and right) of each mouse, once daily for 3 consecutive days. A further group of mice, treated with an equivalent volume of the vehicle, acted as the negative control group. A hair dryer was passed briefly over the ear's surface to prevent loss of any of the test item applied. Five days after the first topical application, all mice were administered 250 µl of 80 µCi/ml 3HTdR (equal to 20.0 µCi) by intravenous injection via a tail vein. Approximately 5 hours later, draining auricular lymph nodes were rapidly excised and pooled for each experimental group (8 nodes/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 twice with phosphate buffered saline, the lymph node cells were resuspended in 5% trichloroacetic acid (approximately 3 ml) and incubated at approximately 4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a beta-scintillation counter as the number of radioactive disintegrations per minute (dpm).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation:
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 = estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferation activity i.e. a Stimulation Index (SI) of 3;
(a, b) and (c, d) = respective co-ordinates of the two pair of data lying immediately below and above the S.I. value of 3 on the local lymph node assay dose reponse plot.

Results and discussion

Positive control results:
No clinical signs were seen in any animal treated with 5% alpha-hexylcinnamaldehyde when compared with the corresponding control group. A slight ear erythema occurred on the second application day at both dosing sites in all mice treated with 10 or 25% concentrations (with the exception of 1 animal), which persisted for the remainder of the in-life phase of the study or for a total of 3 days, separately. On the second application day, a slight ear swelling was observed at both dosing sites in all mice treated with 25%, persisting for the remainder of the in-life phase of the study or for a total of 3 days, separately. The stimulation indices for the 5, 10 and 25% test concentrations were 2.7, 3.4 and 12.4 respectively, showing a clear dose-response relationship. The EC3 (the estimated concentration of test item required to produce a stimulation index of 3) was 7.1% (w/v). As a test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the stimulation index, alpha-hexylcinnamaldehyde responded as expected for a positive control i.e. it was found to be a skin sensitizer.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
2.2
Test group / Remarks:
25%
Parameter:
SI
Value:
1.8
Test group / Remarks:
50%
Parameter:
SI
Value:
4.9
Test group / Remarks:
100%
Parameter:
EC3
Remarks:
% (w/v)
Value:
69.4
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Measured disintegrations per minute: 0, 4337, 9656, 7670, 21259 for the background (1 ml of 5% trichloroacetic acid), control, 25, 50 and 100% groups respectively. Disintegrations per minute per lymph node: 542, 1207, 959 and 2657 for the control, 25, 50 and 100% groups respectively.

Any other information on results incl. tables

No deaths occurred during the study, there were no symptoms of local toxicity at the ears of the animals and no systemic findings during the study period. Body weights, recorded prior to the first application and prior to necropsy, were within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In a local lymph node assay in mice, conducted according to OECD test guideline 429 and in compliance with GLP, tri(isopropyl)silyl acrylate was tested at 25, 50 and 100% and found to be a skin sensitizer (Stimulation Index ≥3 at 100%) with an EC3 value of 69.4%.
Executive summary:

Tri(isopropyl)silyl acrylater was tested in a local lymph node assay, conducted to OECD test guideline 429 and in compliance with GLP.

Test material (25 µl of 25, 50 or 100%

in acetone/olive oil was topically applied to the entire dorsal surface of each ear lobe of groups of 4 female mice, daily for 3 consecutive days. Five days after the first topical application, 3H-methyl thymidine was injected intravenously into a tail vein and, approximately 5 hours later, mice were sacrificed, draining auricular lymph nodes excised, pooled per group and the incorporation of 3H-methyl thymidine measured.

All treated animals survived the study period and no clinical signs of toxicity were observed. The stimulation indices for the 25, 50 and 100% treatment groups were 2.2, 1.8 and 4.9 respectively.

Tri(isopropyl)silyl acrylate was found to be a skin sensitizer (SI 3 at 100%) with an EC3 value of 69.4% under the conditions of the test.