Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: 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

Data source

Reference
Reference Type:
publication
Title:
The influence of perfumes on the sensitising potential of cosmetic bases - I. A technique for evaluating sensitising potential
Author:
Brulos, M.F., Guillot, J.P., Martini, M.C. and Cotte, J.
Year:
1977
Bibliographic source:
J. Soc. Cosmet. Chem. 28 357-365 (1977) Society of Cosmetic Chemists of Great Britain

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzylidene acetone
- Substance type: organic
- Physical state: solid

Test animals

Species:
guinea pig
Strain:
Hartley
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

Test system

Type of coverage:
occlusive
Preparation of test site:
other: clipped
Controls:
other: the untreated skin served as control
Amount / concentration applied:
0.5 g or 0.5 ml (1 % dilution of test substance in ethanol (70%), aqueous lotion, emulsion water in oil and emulsion oil in water)
Duration of treatment / exposure:
48 h
Observation period:
48 h
Number of animals:
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.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: qualitative result
Basis:
mean
Remarks on result:
other: no irritation at the concentrations employed
Irritation parameter:
other: qualitative result
Remarks on result:
other: However, other emulsions (oil in water) gave positive reactions for skin irritation (primary irritation).
Irritant / corrosive response data:
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 (CAS 1896-62-4) 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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: EU-GHS
Conclusions:
The pre-test performed in guinea pigs showed benzylidene acetone not to cause primary irritation. However, other oil-water-emulsions have been shown to cause skin irritation.
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 irritation to skin.