Registration Dossier

Administrative data

Description of key information

1) sensitisation in humans - sensitising
2) sensitisation in guinea-pigs - sensitising
3) sensitisation in guinea pigs - sensitising
4) non-LLNA - sensitising
5) LLNA, OECD 429 - sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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this purpose the LLNA data derived from previously conducted studies were compiled from the published literature and unpublished sources.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.
Statistics:
Not reported.
Key result
Parameter:
EC3
Value:
ca. 3.7

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:
Category 1 (skin sensitising) based on GHS criteria
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) No 1272/2008.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
in vivo
Type of information:
experimental study
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:
no guideline followed
Principles of method if other than guideline:
A technique was developed 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.
GLP compliance:
no
Type of study:
other: development of a technique for determining the sensitising potential of cosmetic products in the albino guinea-pig.
Justification for non-LLNA method:
Old publication where LLNA wasn't standard procedure yet.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Albino Hartley guinea-pigs of both sexes
- Weight at study initiation: 300 - 400 g
- Housing: in an air conditioned animal house in cages of 5, 600 x 540 x 315 mm. Cages have grilled bases to eliminate soiling with faeces, litter, etc.
- Diet (e.g. ad libitum): 50 g of granules per day (granulés Cobaye U.A.R. N° 114). This diet is supplemented with carrots.
- Water (e.g. ad libitum): freely available
- Acclimation period: 14 days

OTHER:
- Duration of the study: 40 days
Route:
intradermal and epicutaneous
Vehicle:
other: undiluted for induction and a 1 % dilution of test substance in ethanol (70%), 1 % in an aqueous lotion, 1 % in an emulsion water in oil and 1 % in an emulsion oil in water for challenge applications
Concentration / amount:
Induction: two intradermic injections of Freund's complete adjuvant and 10 applications of 0.5 g or 0.5 ml of the undiluted test substance under occlusive conditions.
Challenge: 0.5 g or 0.5 ml of the test substance dilution
Route:
epicutaneous, occlusive
Vehicle:
other: undiluted for induction and a 1 % dilution of test substance in ethanol (70%), 1 % in an aqueous lotion, 1 % in an emulsion water in oil and 1 % in an emulsion oil in water for challenge applications
Concentration / amount:
Induction: two intradermic injections of Freund's complete adjuvant and 10 applications of 0.5 g or 0.5 ml of the undiluted test substance under occlusive conditions.
Challenge: 0.5 g or 0.5 ml of the test substance dilution
No. of animals per dose:
20 animals in total / 6 animals for skin irritation tests
Details on study design:
SCREENING FOR PRIMARY IRRITATION
Before commencing the sensitisation study it is essential to check for primary irritation (application of test substance for 48 h under occlusive patch using six guinea-pigs).
All substances producing primary irritation are eliminated unless destined to be used in a diluted form. For these latter cases the minimum dilution which does not cause irritation is determined and used for the challenge application. Induction is always performed using the undiluted test substance.

TEST SITE
- Test site: back of the animals immediately behind the left scapuluni
- Type of wrap if used: occlusive patch (The test substance is covered by an impermeable occlusive patch 22 mm in diameter. This is held in place by a microporous adhesive border 10 mm wide (Neodermotest). The entire patch is covered between treatment times by an elastic sleeve. )

REMOVAL OF TEST SUBSTANCE
- Time point: after 48 hours

SCORING SYSTEM:
Macroscopic cutaneous examination
1, 7, 24 and 48 h after the removal of the occlusive dressing the intensity of the erythematous reaction and eschar formation are evaluated using the following scale:
0 - no erythema
1 - slight erythema (hardly visible)
2 - erythema distinct
3 - erythema moderate to severe
4 - erythema severe (red/purple) with the formation of light eschars (profound lesions)
N.B. - Any other anomaly occurring at the challenge site (eg. papules, vesicles, oedema exfoliation) should also be noted. The induction site must also be unspected in case of any focal reaction (reactivation of induction site). It is extremely important that all readings are performed under the same conditions (particularly the same conditions of lighting).
The results are interpreted by calculating the percentage of guinea-pigs sensitised. The animals counted as positive are those which,
(a) present at least once in four readings a reaction scoring two or more, or
(b) present focal reactions whatever the reaction observed at the challenge site, or
(c) present vesicles.

Expression of results
After the macroscopic cutaneous examination and histological examination have been conducted 'blind', the results are evaluated as shown below.
The result is positive if one or more animals show distinct macroscopic reactions confirmed histologically as sensitisation reactions.
The result is negative if no animal shows a distinct macroscopic reaction or if the histology does not confirm the macroscopic observation.
The result is doubtful if a distinct macroscopic reaction is noted but the histological examination is unable to determine its origin.

Sensitisation in the guinea-pig is induced by intradermal injections of Freund's adjuvant and by topical applications of the test substance under occlusive dressings. After a rest period of 12 days a single challenge application of the test substance, again under an occlusive dressing, provokes the appearance of a sensitisation reaction.
The use of occlusive patches to increase the hydratation of the skin and permeability of the stratum corneum plus the administration of Freund's complete adjuvant to maximize the immunological response gives the technique sufficient sensitivity to detect even weak allergens.
Key result
Reading:
1st reading
Group:
test group
Dose level:
test group treated with 1 % of test substance in ethanol (70 %)
No. with + reactions:
3
Total no. in group:
6
Clinical observations:
Macroskopic: mean of erythema score: 1-81, Histologically: 3/6 showed lesions of allergic type with intense spongiosis, exocytosis and weeping
Remarks on result:
other: see Remark
Remarks:
Group: other: test group treated with 1 % of test substance in ethanol (70 %). Dose level: 1 %. No with. + reactions: 3.0. Total no. in groups: 6.0. Clinical observations: Macroskopic: mean of erythema score: 1-81, Histologically: 3/6 showed lesions of allergic type with intense spongiosis, exocytosis and weeping.
Key result
Reading:
1st reading
Group:
test group
Dose level:
test group treated with 1 % of test substance in aqueous lotion
No. with + reactions:
2
Total no. in group:
3
Clinical observations:
Macroskopic: mean of erythema score: 2-15, Histologically: 2/3 showed reactions of allergic type. One was a major reaction with necrosis and erosion similar to an intolerance of mixed type (caustic and allergic)
Remarks on result:
other: see Remark
Remarks:
Group: other: test group treated with 1 % of test substance in aqueous lotion. Dose level: 1 %. No with. + reactions: 2.0. Total no. in groups: 3.0. Clinical observations: Macroskopic: mean of erythema score: 2-15, Histologically: 2/3 showed reactions of allergic type. One was a major reaction with necrosis and erosion similar to an intolerance of mixed type (caustic and allergic).
Key result
Reading:
1st reading
Group:
test group
Dose level:
test group treated with 1 % of test substance in an emulsion water in oil
No. with + reactions:
3
Total no. in group:
3
Clinical observations:
Macroskopic: mean of erythema score: 1-97, Histologically: 3/3 showed a pathological aspect. In one the inflammatory process was clearly allergic with spongiosis, lymphocyte exocytosis and intense parakeratosis
Remarks on result:
other: see Remark
Remarks:
Group: other: test group treated with 1 % of test substance in an emulsion water in oil. Dose level: 1 %. No with. + reactions: 3.0. Total no. in groups: 3.0. Clinical observations: Macroskopic: mean of erythema score: 1-97, Histologically: 3/3 showed a pathological aspect. In one the inflammatory process was clearly allergic with spongiosis, lymphocyte exocytosis and intense parakeratosis.
Key result
Reading:
1st reading
Group:
test group
Dose level:
test group treated with 1 % of test substance in an emulsion oil in water
No. with + reactions:
2
Total no. in group:
3
Clinical observations:
Macroskopic: mean of erythema score: 2-39, Histologically: 2/3 showed very slight surface parakeratosis with neither spongiosis nor exocytosis. Aspect pathological, but difficult to classify
Remarks on result:
other: see Remark
Remarks:
Group: other: test group treated with 1 % of test substance in an emulsion oil in water. Dose level: 1 %. No with. + reactions: 2.0. Total no. in groups: 3.0. Clinical observations: Macroskopic: mean of erythema score: 2-39, Histologically: 2/3 showed very slight surface parakeratosis with neither spongiosis nor exocytosis. Aspect pathological, but difficult to classify.
Reading:
1st reading
Group:
positive control
Dose level:
2% Paraphenylenediamine
No. with + reactions:
2
Total no. in group:
3
Clinical observations:
Erythema score: 2.16, strong allergic reactions
Reading:
1st reading
Group:
negative control
Remarks on result:
not measured/tested

Benzylideneacetone in an oil/water emulsion gave questionable results, as the macroscopic observations were not confirmed histologically. However, instead of being taken 7 h after the removal of the occlusive dressing, samples were taken 48 h after patch removal (i.e. 96 h after application of test substance). When one examines the macroscopic development of cutaneous reactions one notes a diminution in the intensity of the erythema between 24 and 48 h after the removal of the occlusive dressing. Perhaps in this case the biopsies were conducted too late. For benzylideneacetone the histological examination gave rather indeterminate results for similar reasons. Other studies conducted with other oil/water emulsions containing benzylideneacetone have demonstrated reactions which are of primary irritation in nature. It is therefore important to perform skin biopsies no later than 7 h after the removal of the patch to reduce the incidence of false negative results. Precautions were taken to avoid interference due to irritation.

Table 1
  Test substance Macroscopic examination  
Data at hand Chemical name Concentration Vehicle % animals reacting Mean of erythema score Histological examination Result
Experimentally a strong sensitiser for both man and the guinea-pig Has caused numerous sensitizations at concentrations of 2% (5) Paraphenylene-diamine 2% Ethanol (70°) 100 2-16 2/3 showed allergic type inflammatory reactions with intense spongiosis and massive lymphocyte exocytosis. In one there was necrosis, with erosion, weeping and a squamous crust +
0-5% Ethanol (70°) 93-7 2-20 5/6 showed inflammatory reactions which were clearly allergic with spongiosis +
Experimentally a weak sensitiser for man and the guinea-pig Benzocaine 2% Sterile nentral olive oil 38-8 38-8 1-47 1/2 showed a moderate allergic reaction. The epidermis was slightly thickened. Serous parakeratosis and lymphocyte exocytosis +
Has caused sensitisation at concentrations of 1% (13) 0-5% Sterile nentral olive oil 29-4 1-36 1/3 showed a clearly allergic response with spongiosis and sub-normal vesicles +
No experimentally positive results in animal. Has caused sensitisation in man at concentrations of 5% (14) Butyl parahydroxybenzoate 5% Sterile neutral olive oH 294 1-70 2/6 showed pathological aspects. The worst showed spongiosis, weeping, squamous crust and moist lymphocyte infiltration. Aspect clearly allergic +
No sensitising reactions observed in man in use Shampoo 30%* Water 0 0-15 3/3 showed no allergic response
Experimentally a classHIsensitiser in the guinea-pig and class IV in man (10) (11). Has caused many sensidsations (5) Hydroquinone monobenzyl ether 5% Ethanol (70°) 47 1-48 No pathological aspect was noted (3/3)  
Experimentally sensitising in man at 20% (17) Dihydro-coumarine 1% Ethanol (70°) 57.1 1-82 1/3 gave a pathological picture of allergic type with moist parakeratosis, exocytosis and slight spongiosis. Allergiotype +
Experimentally sensitising in man at 4% (17) Citral 1% Ethanol (70°) 61-1 1-88 3/7 showed an aspect clearly pathological with one distinctly allergic type with spongiosis +
Experimentally a strong sensitiser in man at 2% (17) Benzylidene acetone 1% Ethanol (70°) 66-6 1-81 3/6 showed lesions of allergic type with intense spongiosis, exocytosis and weeping +
1% Aqueous lotion 70-5 2-15 2/3 showed reactions of allergic type. One was a major reaction with necrosis and erosion similar to an intolerance of mixed type (caustic and allergic) +
1% Emulsion water in oil 93-7 1-97 3/3 showed a pathological aspect. In one the inflammatory process was clearly allergic with spongiosis, lymphocyte exocytosis and intense parakeratosis +
1% Emulsion oil in water 100 2-39 2/3 showed very slight surface parakeratosis with neither spongiosis nor exocytosis. Aspect pathological, but difficult to classify Doubtful
* The induction (10 applications was made at 100%).

Of the twelve preparations tested (seven different substances) ten gave good correlation between the macroscopic and histological results.

The 5% hydroquinone monobenzyl ether made up in ethanol (70°) and the benzylideneacetone in an oil/water emulsion gave questionable results, as the macroscopic observations were not confirmed histologically. However, instead of being taken 7 h after the removal of the occlusive dressing, samples were taken 48 h after patch removal (i.e. 96 h after application of test substance). When one examines the macroscopic development of cutaneous reactions one notes a diminution in the intensity of the erythema between 24 and 48 h after the removal of the occlusive dressing. Perhaps in this case the biopsies were conducted too late. For benzylideneacetone the histological examination gave rather indeterminate results for similar reasons. Other studies conducted with other oil/water emulsions containing benzylideneacetone have demonstrated reactions which are of primary irritation in nature. It is therefore important to perform skin biopsies no later than 7 h after the removal of the patch to reduce the incidence of false negative results. Precautions were taken to avoid interference due to irritation. However, even in healthy areas, the skin of sensitised subjects is more susceptible to irritation than the skin of normal subjects. Furthermore the use of Freund's adjuvant may lead to the appearance of non-specific reactions.

It must also be remembered that the margin between the maximum non-irritant concentration and the minimum necessary to cause a sensitisation reaction is much smaller in the guinea-pig than in man. This is why it seems necessary to consider only those macroscopic reactions scoring two or more which also reduces the need to conduct a histological examination. We abandoned the Magnusson and Kligman system for the expression of results. Cosmetic products ready for commercialization are unlikely to contain strong sensitisers since known sensitisers are eliminated from their composition. Unlike a pharmaceutical product where the therapeutic activity may be great enough to permit the use of a component with doubtful sensitising potential, uncertainty is never acceptable for a cosmetic product. Thus it is important to be clear about the expression of results during the animal test (the more so since extrapolation to man entails many approximations). It is possible to use this technique for detecting the sensitizing potential of cosmetic bases (aqueous or oil solutions, and water/oil or oil/water emulsions) as well as the potential of the perfume content or the combined mixture.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Benzylideneacetone in ethanol, in aqueous lotion and in an emulsion water/oil gave clear positive results. Benzylideneacetone in an oil/water emulsion gave questionable results, as the macroscopic observations were not confirmed histologically.
Executive summary:

The skin irritation 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 sensitizers. 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.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: publication, which meets basic scientific principles, also published in the peer-rewieved handbook Fragrance Raw materials monographs
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
This publication 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.
GLP compliance:
no
Type of study:
patch test
Justification for non-LLNA method:
Old publication where LLNA wasn't standard procedure yet.
Species:
human
Strain:
other: not applicable
Sex:
not specified
Details on test animals and environmental conditions:
25 volunteers
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
2 %
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
2 %
No. of animals per dose:
25 human volunteers
Details on study design:
No details given, other than according to the maximisation test of Kligman (1966).
Challenge controls:
No details given, other than according to the maximisation test of Kligman (1966).
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Group:
test group
Dose level:
2 %
No. with + reactions:
12
Total no. in group:
25
Clinical observations:
sensitisation
Remarks on result:
other: Reading: other: not given. Group: test group. Dose level: 2 %. No with. + reactions: 12.0. Total no. in groups: 25.0. Clinical observations: sensitisation.
Group:
negative control
Remarks on result:
not measured/tested
Group:
positive control
Remarks on result:
not measured/tested
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
This publication 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.
Executive summary:

This publication 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.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
in vivo
Type of information:
experimental study
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
Principles of method if other than guideline:
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 benzylideneacetone (known allergenic activity), a reference sensitiser, which was added to the excipients at the same concentration as the perfumes.
GLP compliance:
no
Type of study:
not specified
Justification for non-LLNA method:
Old publication where LLNA wasn't standard procedure yet.
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
no details given
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
1 % benzylidene acetone was added to the bases
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
1 % benzylidene acetone was added to the bases
No. of animals per dose:
15 animals per group
Details on study design:
Sensitisation in the guinea-pig is induced by intradermal injections of Freund's adjuvant and by topical applications of test substances under occlusive dressings. After a rest period of 12 days a single challenge application of the test substance, again under an occlusive dressing, provokes the appearance of a sensitisation reaction.
Readings were conducted immediately after the removal of the patch in order to eliminate errors due to subsequent scratching, etc. and then, one hour, 24 h and 48 h after removal of the patch.
The last reading at 48 h was accompanied if necessary by a skin biopsy for eventual histological examination. It is however, preferable to perform the biopsy 7 h after the removal of the patch. The erythema was scored from 0 to 4 and any other anomalies ( papules, vesicles, exfoliations) or a reactivation of the induction site were noted.
The number of animals showing an evident reaction (equal to or greater than 2) at any of the readings was calculated and the mean erythema value for the whole group was also determined.
The aim of the histological examination was to determine the allergic characteristics of the reaction. In fact when the macroscopic examination was positive, a histological examination was also conducted. When this examination was positive, sensitization was confirmed. If this examination was negative, for instance, if the reaction noted was of primary irritation, then the overall result was said to be negative. When the macroscopic examination was negative, the histological examination was not conducted.
The test was called 'doubtful' when the macroscopic examination was positive and the histological examination was unable to determine the type of reaction present.
Positive control substance(s):
yes
Remarks:
benzylidene acetone (reference sensitiser)
Key result
Reading:
1st reading
Group:
test group
Dose level:
Lotion with 1% benzylidenacetone
No. with + reactions:
7
Total no. in group:
8
Clinical observations:
mixed allergic and caustic reaction
Remarks on result:
other: Group: other: Lotion with 1% benzylidenacetone. No with. + reactions: 7.0. Total no. in groups: 8.0. Clinical observations: mixed allergic and caustic reaction.
Key result
Reading:
1st reading
Group:
test group
Dose level:
Cream O/W B with 1 % benzylideneacetone
No. with + reactions:
9
Total no. in group:
9
Clinical observations:
moderate orthoergy
Remarks on result:
other: Group: other: Cream O/W B with 1 % benzylideneacetone. No with. + reactions: 9.0. Total no. in groups: 9.0. Clinical observations: moderate orthoergy.
Key result
Reading:
1st reading
Group:
test group
Dose level:
Cream O/W C with 1 % benzylideneacetone
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
moderate orthoergy
Remarks on result:
other: Group: other: Cream O/W C with 1 % benzylideneacetone. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: moderate orthoergy.
Key result
Reading:
1st reading
Group:
test group
Dose level:
Cream W/O D with 1 % benzylideneacetone
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
marked orthoergy
Remarks on result:
other: Group: other: Cream W/O D with 1 % benzylideneacetone. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: marked orthoergy.
Key result
Reading:
1st reading
Group:
test group
Dose level:
Cream W/O E with 1 % benzylideneacetone
No. with + reactions:
7
Total no. in group:
7
Clinical observations:
Allergy + orthoergy
Remarks on result:
other: Group: other: Cream W/O E with 1 % benzylideneacetone. No with. + reactions: 7.0. Total no. in groups: 7.0. Clinical observations: Allergy + orthoergy.
Reading:
1st reading
Group:
negative control
Dose level:
Lotion A without perfume or kown sensitiser
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
no reaction
Reading:
1st reading
Group:
positive control
Dose level:
Benzylideneacetone ethanol 70%
No. with + reactions:
7
Total no. in group:
11
Clinical observations:
Major allergy in three animals
Reading:
1st reading
Group:
negative control
Dose level:
Cream O/W A
No. with + reactions:
2
Total no. in group:
9
Clinical observations:
Moderate primary irritation in two animals
Reading:
1st reading
Group:
negative control
Dose level:
Cream O/W B
No. with + reactions:
1
Total no. in group:
13
Clinical observations:
Minor allergic response in one animal
Reading:
2nd reading
Group:
negative control
Dose level:
Cream O/W C
No. with + reactions:
5
Total no. in group:
13
Clinical observations:
Major allergy in one animal
Reading:
1st reading
Group:
negative control
Dose level:
Cream W/O D
No. with + reactions:
0
Total no. in group:
17
Clinical observations:
No reaction noted
Reading:
1st reading
Group:
negative control
Dose level:
Cream W/O E
No. with + reactions:
0
Total no. in group:
11
Clinical observations:
No reaction noted.

when 1% benzylideneacetone was added to the bases, primary cutaneous irritation reactions were noted in all animals. In order to demonstrate the sensitising potential of benzylideneacetone without the problems of irritation it would be necessary to conduct a challenge reaction with a diluted solution. The intensity of the erythema observed at the end of the sensitisation test was always clearly greater than that observed after the first application even though, as will be seen later, irritation phenomena were dominant.

Table III. Influence of the addition of a known sensitiser (1% benzylidenacetone) to cosmetic bases
Bases   Number of animals showing reactions Mean erythemas per group Macroscopic examination (interpretation) Histological examination
Number of animals examined Reactions Interpretation Conclusion
Lotion  —A— 7/8 2-34 + 3 Mixed allergic and caustic reaction + +
Cream O/W  —A— not performed
Cream O/W  —B— 9/9 2-22 + 4 Moderate orthoergy - -
Cream O/W  —C— 5/5 2-47 + 3 Moderate orthoergy - -
Cream W/O  —D— 10/10 2-67 + 4 Marked orthoergy - -
Cream W/O  —E— 7/7 2-24 + 3 Allergy + orthoergy + +
TableIV.Evaluation of the sensitising potential for the perfume base no.602 127, tested at 1%in ethyl alcohol70%v/v
Product Number of animals showing reactions Mean of erythemas per group Macroscopic examination (interpretation) Histological examination
Number of animals examined Reactions Interpretation Conclusion
No.602 127 (1%in70%v/v ethyl alcohol) 0/12 0-47 - 6 No reaction noted - -

Table V.Influence of addition of perfumes to cosmetic bases
Test substances Number of animals showing reactions Mean of erythemas per group Histological examination
Macroscopic examination (interpretation) Number of animals examined Reactions Inter-pretation Conclusion
4.1/Lotion —A —
Lotion only 1/10 013 - 6 No reaction noted - -
Lotion+perfume
No.602 123 0/12 002 - 3 No reaction noted - -
4.2/Creams O/W
Cream O/W —A— 2/9 0-63 + 3 Moderate orthoergy two animals
+perfume602 127 0/12 0-27 - 3 Normal - -
+perfume602 125 1/11 0-25 + 3 Normal - -
Cream O/W —B— 1/13 0-60 + 6 Slight allergy? response (one animal) - -
+perfume602 127 2/12 0-27 + 3 Normal - -
+perfume602 124 2/13 0-38 + 3 Congested dermis (two animals) - -
Cream O/W —C— 5/13 0-75 + 6 Major allergy (one animal) + +
+perfume602 127 0/11 015 3 Normal - -
+perfume602 123 0/10 010 3 Desquamation in two animals - -
4.3/Creams W/O
Cream W/O —D— 0/14 0-33 - 6 No reaction noted - -
+perfume602 127 1/13 0-38 + 3 No reaction noted - -
+perfume602 125 1/14 0-28 + 3 No reaction noted - -
Cream W/O —E— 0/11 0-56 - 6 No reaction noted - -
+perfume602 127 3/14 0-62 - 3 No reaction noted - -
+perfume602 124 2/12 0-24 + 3 One doubtful reaction   -

The results of these experiments with six excipients which were used as bases for four different aromatic components how that whatever the excipient used the number of animals showing reactions was always very small. In addition, the mean erythema scores were comparable and always inferior to 1(1 = erythema barely visible). Only one result seemed difficult to interpret - creamC : test positive with perfume and negative without perfume. It seems necessary to conduct a new series of tests for this cream. The cutaneous tolerance showed little or no modification due to the perfumes (basic composition or elaborated compositions) with relation to the excipient used. It should be emphasized however, that whenever a possible allergic type reaction was found for a base, or a perfume, or a mixture of the two this conclusion was based, in the majority of cases, on the effects found in one animal only. In addition, in all cases where there was an increase in the reaction score the mean of the erythema for the whole group always remained well below 1 whereas a test substance which is clearly irritant or sensitising is normally around 2.5 (erythema clearly visible).

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Benzylideneacetone acts as reference sensitiser in the report and showed clear sensitising properties.
Executive summary:

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 benzylideneacetone (known allergenic activity), a reference sensitiser, which was added to the excipients at the same concentration as the perfumes.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
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 investigations described here 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 sensitization and 7 of which are believed not to be associated with any significant evidence of allergic contact dermatitis in humans.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Sources of CBA/J strain:
1) Procter and Gamble Company, Miami Valley Laboratories, Cinicinnati, OH
2) Harlan Sprague Dawley, Inc., Indianapolis, IN
3 ) Jackson Laboratories, Bar Harbor, ME
- Sources of CBA/Ca strain:
1) AstraZeneca Central Toxicology Laboratory, Macclesfield, Cheshire, UK
2) Unilever Safety and Environmental Assurance Centre Toxicology Group, Unilever, Sharnbrook, Bedfordshire, UK
3) Harlan Olac, Bicester, Oxfordshire, UK
- Age at study initiation: 6 - 12 weeks
- housed, fed, and handled in compliance with standards set forth by the US Animal Welfare

IN-LIFE DATES: The groups of mice were treated once a day for 3 consecutive days with 25 ml of the test substance.
5 days after the initial treatment, all mice were injected intravenously, via the tail vein, with 250 ml of phosphate buffered saline (PBS) containing [3H]-TdR.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10% w/v in acetone and olive oil (4:1)
25% w/v in acetone and olive oil (4:1)
50% w/v in acetone and olive oil (4:1)
No. of animals per dose:
5 (for benzylidene acetone, Laboratory A)
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
A standard local lymph node assay (LLNA) protocol was used by the laboratories. One laboratory employed the standard protocol and lymph nodes were pooled for individual mice rather than from experimental groups. The robustness of the LLNA has been demonstrated previously that such minor procedural modifications, i.e., using lymph nodes pooled for experimental groups versus nodes from individual animals, are without consequences to the test’s performance. Briefly, groups of mice (n=5, Laboratory A; n=4, Laboratories B and C) were treated once a day for 3 consecutive days on the dorsum of both ears with 25 ml of 1 of 3 concentrations of test material or with vehicle alone.
5 days after the initial treatment, all mice were injected intravenously, via the tail vein, with 250 ml of phosphate buffered saline (PBS) containing 20 µCi of [3H]-methylthymidine. 5 h later, the mice were euthanised and the draining auricular lymph nodes were removed and pooled for each individual mouse (Laboratory A) or for each experimental group (Laboratories B and C). Single cell suspensions were prepared by gentle mechanical disaggregation through either nylon mesh (100 mm pore size; Laboratory A) or 200-mesh stainless steel gauze (Laboratories B and C). Cell suspensions were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) and left at 4°C overnight. The samples were then centrifuged, resuspended in 1 ml 5% TCA and transferred to 10 ml of scintillation cocktail (Laboratory A, Ecolume, ICN Radio chemicals, Irvine, CA; Laboratories B and C, Optiphase MP, LKB Scintillation Products, FSA Laboratory Supplies, UK).
[3H]-TdR incorporation was measured by beta-scintillation counting and expressed as disintegrations per min (dpm) per individual mouse (Laboratory A) or as dpm per node for each experimental group (Laboratories B and C). Stimulation indices (SI) for each experimental group were determined as the increase in [3H]-TdR incorporation relative to the concurrent vehicle treated control group. A material is considered to be positive in the LLNA if a 3-fold or greater increase in proliferation, compared with the concurrent vehicle control, is obtained at 1 or more test concentrations.
Positive control substance(s):
not specified
Statistics:
no data
Positive control results:
no data
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: AOO: 595 +/- 185 10% w/v: 5059 +/- 773 25% w/v: 8070 +/- 352 50% w/v: 7619 +/- 892
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
AOO (vehicle)
Key result
Parameter:
SI
Value:
8.5
Test group / Remarks:
10% w/v
Key result
Parameter:
SI
Value:
13.6
Test group / Remarks:
25% w/v
Key result
Parameter:
SI
Value:
12.8
Test group / Remarks:
50% w/v
Local lymph node assay results  
Chemical and testing lab Vehicleaand concentrations dpm/node or *mean dpm (±SEM) Stimulation index Sensitization classification by LLNA  Human sensitization classification
benzylidene acetone AOO *595±185 1    
  10% w/v 5059±773 8.5  +       +     
Lab A 25% w/v 8070 ±352 13.6
  50% w/v 7619±892 12.8

AOO = acetone: olive oil, 4:1; SEM = Standard error of the mean

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test substance is classified as human skin sensitiser by LLNA.
Executive summary:

The investigations described here were designed to explore further the ability of the LLNA to identify accurately those chemicals that cause allergic contact dermatitis in humans (Ryan, 2000). 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 sensitization 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-sensitizers, 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 sensitizers from those chemicals which do not possess a significant skin sensitization 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.

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) No 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.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin irritation:

According to the 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).