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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
other: Compilation of Historical Local Lymph Node Data
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well-documented publication, which meets basic scientific principles
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
not specified
Principles of method if other than guideline:
The objective was to provide a database of robust in vivo data to calibrate, evaluate, and eventually validate new approaches for skin sensitization testing. For this purpose the LLNA data derived from previously conducted studies were compiled from the published literature and unpublished sources.
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7 - 12 weeks

IN-LIFE DATES: 5 days after exposure, the test animals were sacrified after injection of around 250 µL of phosphate-buffered saline including 20 µCi of tritiated trimidine via the tail vail.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10 % in acetone and olive oil (4:1)
25 % in acetone and olive oil (4:1)
50 % in acetone and olive oil (4:1)
quantity always 25 µl
No. of animals per dose:
no details given
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The LLNA was conducted as described in the literature. Briefly, groups of CBA female mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25 µL of test material or to an equal volume of the relevant vehicle alone. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate-buffered saline containing 20 µCi of tritiated thymidine. The mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute. An SI was calculated for each chemical-treated group as the ratio of the disintegrations per minute in the treated group (or mean disintegrations per minute when individual animals were assessed) to the disintegrations per minute or mean disintegrations per minute of the concurrent vehicle control group. LLNA methodology assesses skin sensitization, not photosensitization.

A database is provided, that comprises LLNA data on 211 individual chemicals. This extensive chemical data set encompasses both the chemical and biologic diversity of known chemical allergens. To cover the range of relative allergenic potencies, the data set includes data on 13 extreme, 21 strong, 69 moderate, and 66 weak contact allergens, classified according to each allergen's mathematically estimated concentration of chemical required to induce a threefold stimulation index. In addition, there are also 42 chemicals that are considered to be non-sensitizers. In terms of chemical diversity, the database contains data pertaining to the chemical classes represented by aldehydes, ketones, aromatic amines, quinones, and acrylates, as well as compounds that have different reactivity mechanisms. In addition, the physicochemical parameters log Kp, log Ko/w and molecular weight are included.

Conclusions: The list of chemicals contained in the data set represents both the chemical and biologic diversity that is known to exist for chemical allergens and non-allergens. It is anticipated that this database will help accelerate the development, evaluation, and eventual validation of new approaches to skin sensitization assessment.

Table 3 - Chemicals, Physicochemical Parameters, LLNA Data, and Potency Categorization

Chemical Structure CAS-No.  Log Kp Log Kow MW Vehicle LLNA
%
LLNA
%
LLNA
%
LLNA
%
LLNA
%
LLNA
%
LLNA
SI%
LLNA
SI%
LLNA
SI%
LLNA
SI%
LLNA
SI%
LLNA
SI%
LLNA
EC3* (%)
Potency Category Reference
Benzylidene acetone
(4-phenyl-3-buten-2-one)
122-57-6 -1,81 2.45 146.19 AOO 10.0 25.0 50.0 - - - 8.5 13.6 12.8 - - - 3.7* Moderate Ryan CA et al.
AOO = acetone and olive oil (4:1); CAS - Chemical Abstract Service number; DEP = diethyl phthalate; dH20 - distilled water; DMF = dimethylformamide; DMSO = dimethyl sulfoxide; EtOH - ethanol; FEMA — Flavor and Extract Manufacturers* Association; Kow = octano-water partition coefficient (log scale); Kp = skin penetration coefficient (log scale); LLNA = local lymph node assay; MEK = methyl ethyl ketone; MW = molecular weight; NC = not calculated; P&G = Procter & Gamble Co.; RIFM = Research Institute for Fragrance Materials.
' Mathematically estimated concentration of test chemical necessary to induce a threefold stimulation index (Sl).
* Value estimated.                      

In this article, the compilation of an extensive chemical data set that embraces a range of chemicals and skin-sensitizing activity is described. All materials have been evaluated through LLNA; for some chemicals, it has been demonstrated that the LLNA EC3 value correlates closely with what is known of the chemical's relative ability to induce sensitization in humans. These data provide a unique and valuable list of chemicals for which the sensitivity, selectivity, and overall accuracy of proposed alternative methods for skin sensitization can be judged. This compilation also provides an invaluable source of data with which to explore other issues, such as the relationship between chemical categorization (on the basis of mechanism of action) and potency. It could also help to define the applicability domain of the LLNA as well as that of existing alternative methodologies and those in development.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: EU-GHS
Conclusions:
As the chemical was identified in an LLNA to bear the potential to act as a moderate skin sensitizer, it has to be classified as sensitising to the skin and requires classification and labelling.
Executive summary:

A database is provided, that comprises LLNA data on 211 individual chemicals (Gerberik, 2005). However, the LLNA for benzylidene acetone was conducted as described in the literature. Briefly, groups of CBA female mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25 µL of test material or to an equal volume of the relevant vehicle alone. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate-buffered saline containing 20 µCi of tritiated thymidine. The mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The chemical was identified to bear the potential to act as a moderate skin sensitizer and has to be classified as sensitising to the skin and requires classification and labelling according to regulation (EC) 1272/2008.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test item is also characterised in a publication, which lists the characteristics of benzylidene acetone, which include it to be a sensitiser in humans (Maximisation test) and a skin irritant in rabbits and humans (Opdyke, 1973, Fragrance raw materials monographs - benzylidene acetone).

The skin irritation / sensitisation potential of benzylidene acetone was evaluated in guinea pigs (Brulos, 1977). The aim of the study was however the development of a test for determining the sensitising potential of cosmetic products in the albino guinea-pig. It consists of the administration of Freund's complete adjuvant by intradermal injection and subsequent application of the test substance topically using an occlusive patch. The technique is therefore particularly well suited for the testing of finished products. Additionally the skin irritation of the substances was tested in a pre-test. The complete method described here gave good results for the detection of weak sensitisers. Since this test avoids the use of an increase in concentration to maximize the reaction nor does it make use of intradermal injections as the route of administration it seems particularly well adapted to the testing of finished products whatever their form. Concerning skin irritation benzylidene acetone showed no skin irritation at the concentrations employed (1 % in ethanol, 1 % in aqueous lotion, 1 % in a water-in-oil emulsion, 1% in a oil-in-water emulsion). However, other oil-in-water emulsions have been shown to cause primary irritation. Therefore the test item has to be considered to be irritant to skin. Concerning skin sensitisation, benzylidene acetone was shown to induce a positive result for the dilutions in ethanol, in aqueous solution and in the emulsion water in oil. The result after application of the test substance in an emulsion oil-in-water was doubtful. In conclusion, the test substance has to be considered to be a skin sensitiser.

The sensitising potential of benzylidene acetone was characterised in this publication (Rochas, 1977). A sensitisation test (devised for use with finished cosmetic preparations for which the Magnusson-Kligman test is not entirely suitable) was used to test a series of cosmetic formulations with and without the addition of perfumes. The reactions obtained were examined and compared with those caused by benzylidene acetone (known allergenic activity), a reference sensitiser, which was added to the excipients at the same concentration as the perfumes.

A database is provided, that comprises LLNA data on 211 individual chemicals (Gerberik, 2005). However, the LLNA for benzylidene acetone was conducted as described in the literature. Briefly, groups of CBA female mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25 µL of test material or to an equal volume of the relevant vehicle alone. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate-buffered saline containing 20 µCi of tritiated thymidine. The mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The chemical was identified to bear the potential to act as a moderate skin sensitiser and has to be classified as sensitising to the skin and requires classification and labelling according to regulation (EC) 1272/2008.

The investigations described by Ryan and coworkers (Ryan, 2000) were designed to explore further the ability of the LLNA to identify accurately those chemicals that cause allergic contact dermatitis in humans. Measures amongst 3 independent laboratories have been performed. LLNA responses induced by a total of 18 test chemicals were considered, 11 of which are known to cause skin sensitisation and 7 of which are believed not to be associated with any significant evidence of allergic contact dermatitis in humans. The LLNA correctly classified 16 of the 18 materials. The 11 chemicals tested which are associated with allergic contact dermatitis in humans were found to be positive in the LLNA. Of the 7 materials believed to be non-sensitisers, 5 were negative in the LLNA and 2 produced positive results. Collectively, these data provide additional evidence that the LLNA is able to discriminate skin sensitisers from those chemicals which do not possess a significant skin sensitisation potential and thus provides a method for hazard identification that offers important animal welfare benefits. The test substance benzylidene acetone (4 -phenyl-3 -buten-2 -one) is classified as human skin sensitiser by LLNA.

 


Migrated from Short description of key information:
1) sensitisation in humans - sensitising
2) sensitisation in guinea-pigs - sensitising
3) sensitisation in guinea pigs - sensitising
4) LLNA - sensitising
5) LLNA - sensitising

Justification for classification or non-classification

Skin irritation:

According to the European regulation (EC) No. 1272/2008, the test material does meet the criteria for classification and will require labelling as a skin sensitiser (Cat. 1, H317).