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Administrative data

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 6-12 weeks
- Housing: under standard conditions
- Diet: ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

No further data
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test substance and positive control: 1, 2.5, 5, 10, 25 and 50%
No. of animals per dose:
4
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: A stimulation index (SI) relative to the concurrent vehicle treated control value was calculated for each group. The criterion for a positve response is that one or more concentrations of the test substance should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance daily for 3 consecutive days. Five days after initiation of exposure an injection of 250 µL phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine (³HTdR) was made into the tail vein of each experimental mouse. Five hours later, following injection of ³HTdR, the mice were sacrificed and draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension was prepared by gentle separation through a 200 mesh stainless steel gauze. The cell suspensions were washed two times with an excess of PBS and precipitated with 5% trichloroacetic acid at 4 °C for 12 h. The pellets were resuspended in 1 mL of 5% trichloroacetic acid and transferred to 10 mL of scintillation fluid.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The EC3 value was calculated by interpolating between 2 points on the SI axis, one immediately above, and the other immediately below, the SI value of 3. The vehicle-treated control (SI=1) cannot be used for the latter. Where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a, b) and (c, d) respectively, then the EC3 value may be calculated using the following equation: EC3 = c + [(3 - d)/(b - d)] × (a - c)
Positive control results:
The EC3 value calculated for the positive control was 14.7.
Key result
Parameter:
SI
Value:
0.73
Test group / Remarks:
1% (w/v)
Key result
Parameter:
SI
Value:
1.76
Test group / Remarks:
2.5% (w/v)
Key result
Parameter:
SI
Value:
7.84
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
8.76
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
18.96
Test group / Remarks:
25% (w/v)
Key result
Parameter:
EC3
Value:
3
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
185
Test group / Remarks:
1% (w/v)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
450
Test group / Remarks:
2.5% (w/v)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
2 000
Test group / Remarks:
5% (w/v)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
2 233
Test group / Remarks:
10% (w/v)
Key result
Parameter:
other: disintegrations per minute (DPM)
Value:
4 834
Test group / Remarks:
25% (w/v)
Interpretation of results:
other: Skin Sens Cat 1B is required according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the mouse Local Lymph Node Assay the test substance revealed a SI ≥ 3.0 at concentrations of 5, 10 and 25%. The EC3 value was 3.0%. Therefore, the test substance is considered as a moderate sensitiser.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitising potential of the test substance was evaluated in a LLNA test according to OECD 429 (2001).The study was conducted on four female mice per dose group (1, 2.5, 5, 10 and 25% w/v). Each animal received 25 µL of the substance to the dorsum of each ear. Animals were treated once daily for three consecutive days. Five days after initiation of exposure, all animals were injected with tritiated methyl-thymidine in the tail vein. Five hours later, animals were sacrificed, and the draining auricular lymph nodes removed and prepared for cell suspension and scintillation counting. A vehicle control (4:1 v/v acetone:olive oil) group was run concurrently. The test substance produced stimulation indices of 0.73, 1.76, 7.84, 8.76 and 18.96 at concentrations of 1, 2.5, 5, 10 and 25% (w/v) in 4:1 v/v acetone:olive oil, respectively and a EC3 value of 3.0% was calculated. Therefore the test substance was considered to be a moderate sensitiser.

Two additional publications are available (Klecak et al., 1977 and Ryan et al., 2000; not included as robust study summary in IUCLID) showing sensitising properties of the test substance. Klecak et al. summarises four different studies in guinea pigs (open epicutaneous test, Draize test, maximisation test and an intradermal test with Freund´s complete adjuvant) all showing sensitising properties of the test substance at different concentrations and undiluted, repectively (1977). In the OET a minimum sensitizing concentration of 0.3% and a minimum eliciting concentration of 0.03% were determined. Ryan et al. showed sensitising properties of the test substance in an murine local lymph node assay where stimulation indices of > 3 were produced at all tested concentrations (25, 50 and 100% (w/v) in acetone:olive oil) (2000).

In conclusion, the test substance is considered to be a weak skin sensitiser with regard to the criteria of the CLP regulation (Skin Sens. 1B).

References:

Klecak, G. et al. (1977): Screening of fragrance materials for allergenicity in the guinea pig. Journal of the Society of Cosmetic Chemists, Vol. 28, pp. 53 - 64

Ryan, C. A. et al. (2000): Activity of human contact allergens in the murine local lymph node assay. Contact Dermatitis, Vol. 43, pp. 95 - 102

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance meet the criteria for classification as Skin Sens 1B (H317) according to Regulation (EC) No 1272/2008.