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Description of key information

Skin sensitisation (OECD 429): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Feb - 05 Mar 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in 2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Federal Office of Public Health, the Swiss Agency for the Environment, Forests and Landscape and the Intercantonal Office for the Control of Medicines, Bern, Switzerland
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Jlbm
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Füllinsdorf, Switzerland
- Age at study initiation: 8 - 12 weeks old
- Weight at study initiation: 18.2 - 22.1 g
- Housing: in groups of 4 in Makrolon cages (type-3), softwood was used as bedding material
- Diet: pelleted standard Kliba 3433, mouse maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: approx. 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30-70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50% (w/v)
No. of animals per dose:
range finding study: 2 females
main study: 4 females
Details on study design:
RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test substance concentration, a non-GLP pretest was performed in two animals with four concentrations (10%, 25% and 50% (w/v) and 100%).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A test substance is regarded as a sensitizer if two criteria are fulfilled. First, the exposure to at least one concentration of the test substance resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control animals, as indicated by the Stimulation Index (SI). Second, the data must be 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: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration. The application was repeated on Days 2 and 3. Five days after the first topical application, each experimental mouse was administered 250 µl of 85.3 µCi/mL 3H-methyl thymidine (3H-TdR) by intravenous injection via the tail vein. Approximately five hours later, the draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions of pooled lymph node cells were prepared in PBS by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). Pooled lymph node cells were washed twice with PBS, resuspended in 5% trichloroacetic acid and incubated at approx. 4 °C for at least 18 h for precipitation of macromolecules.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The results of a reliability test with three concentrations of hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v) was performed not more than 6 months previously (Jan 2003) in CBA/CaOlaHsd mice using the same materials and essential procedures. The SI values calculated for the substance concentrations 5, 10 and 25% were 2.5, 3.7 and 9.7 respectively. An EC3 value of 7.08% was calculated.
Parameter:
SI
Value:
9.8
Test group / Remarks:
10%
Parameter:
SI
Value:
12.2
Test group / Remarks:
25%
Parameter:
SI
Value:
12.3
Test group / Remarks:
50%
Parameter:
EC3
Value:
1.56
Remarks on result:
other:
Remarks:
calculated based on ICCVAM method; see "Any other information on results incl. tables

Table1: Radioactivity counts (DPM) and Stimulation Index (SI).

Group

TS1(%)

DPM2/lymph node

SI

1

0

578

-

2

10

5647

9.8

3

25

7068

12.2

4

50

7113

12.3

1TS = test substance (% w/v)

2DPM= Disintegrations per minute

Viability/Mortality: No deaths occurred during the study period.

Clinical signs: No clinical signs were observed in any animals of the control group. On the second application day, a slight to moderate swelling was observed at both dosing sites in all animals treated with test substance concentrations 10% and 25%, persisting for the remaining days. About 1.5 h after the first topical application, a slight swelling was observed at both dosing sites in all animals treated with test substance concentration 50%. On the second application day, the swelling increased to severe and a slight erythema was observed at both dosing sites. The signs persisted for the remaining days.

Body weights: The body weight of treated animals was within the range of the control.

EC3 value: The EC3 value was not calculated in the study report. However, it is possible to extrapolate the EC3 value using an equation provided by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) in which the two lowest test concentrations from the dose-response curve are used, provided the lowest SI value approaches the value of 3 and that a linear dose-response exists. The equation is as follows:

EC3 = 2 ^ [log2(c) + ((3 -d)/(b-d)) x (log2(a) - log2(c))], where a = dose concentration for next to lowest SI above 3; b = next to lowest SI above 3; c = dose concentration for lowest SI above 3; d = lowest SI above 3.

According to this extrapolation the EC3 value can be estimated as 1.56.

Interpretation of results:
other: CLP/EU GHS Category 1A (H317) according to Regulation (EC) No 1272/2008
Conclusions:
CLP: Skin sens 1A, H317
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitising potential of the test substance was investigated in a Local Lymph Node Assay (LLNA) in mice according to OECD Guideline 429 and in compliance with GLP (Wang-Fan, 2003). Based on the results of a pre-screen test, test substance concentrations of 10%, 25% and 50% (w/v) in acetone/olive oil (4:1 v/v) were selected for the treatment of mice in the main study. In this experiment, 4 female CBA/Jlbm mice per test group were treated with the test substance or vehicle acetone/olive oil (4:1 v/v) alone for three consecutive days by topical application on the ears (25 µL/ear). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each experimental group. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The DPM/lymph node values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 5647, 7068 and 7113 DPM respectively. The DPM/lymph node value for the vehicle control group was 578 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 9.8, 12.2 and 12.3 respectively. The EC3 value was not calculated in the study report. However, it is possible to extrapolate the EC3 value using an equation provided by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) in which the two lowest test concentrations from the dose-response curve are used, provided the lowest SI value approaches the value of 3 and that a linear dose-response exists. According to this extrapolation the EC3 value can be estimated as 1.56. Clinical observations revealed a slight to moderate swelling at both dosing sites in all animals treated with test substance concentrations 10% and 25% on the second application day, persisting for the remaining days. In addition, a slight swelling was observed at both dosing sites in all animals treated with test substance concentration 50% at about 1.5 h after the first topical application. The swelling increased to severe and a slight erythema at both dosing sites on the second application day, persisting for the remaining days. The six-month reliability check with hexyl cinnamic aldehyde indicated that the LLNA is an appropriate model for testing for contact hypersensitivity. Based on the results, the test substance was regarded as a skin sensitizer under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance is classified as Skin Sensitiser Category 1A according to Regulation (EC) 1272/2008.