Registration Dossier

Administrative data

Description of key information

1) Skin irritation in guinea-pigs - irritating
2) Skin irritation in rabbits - irritating
3) Skin irritation in humans - not irritating in a 2 % solution in petrolatum
4) Eye irritation in rabbits, according to OECD 405 and EU method B.5 - not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

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
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
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
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.
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).
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not measured/tested
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.

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 2 (irritant) based on GHS criteria
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.

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: 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
Species:
human
Strain:
other: not applicable
Details on test animals and environmental conditions:
no details given, as the primary publication is not available
Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
other: 2 % concentration in petrolatum
Controls:
other: the untreated skin served as control
Amount / concentration applied:
not given
Duration of treatment / exposure:
not given
Observation period:
not given
Details on study design:
Tested in a 2 % concentration in petrolatum it produced no irritation in a 48 hr closed-patch test in 25 human subjects (Kligman, 1972).
Irritation parameter:
other: qualitative result
Remarks on result:
other: Tested in a 2 % concentration in petrolatum it produced no irritation in a 48 hr closed-patch test in 25 human subjects (Kligman, 1972).
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritant / corrosive response data:
Tested in a 2 % concentration in petrolatum it produced no irritation in a 48 hr closed-patch test in 25 human subjects (Kligman, 1972).
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
Tested in a 2 % concentration in petrolatum it produced no irritation in a 48 hr closed-patch test in 25 human subjects (Kligman, 1972).
Executive summary:

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 not irritating in humans (Opdyke, 1973, Fragrance raw materials monographs - benzylidene acetone).

In there it is stated that Benzylidene acetone tested in a 2 % concentration in petrolatum produced no irritation in a 48 hr closed-patch test in 25 human subjects (Kligman, 1972).

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: 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
Species:
rabbit
Strain:
not specified
Details on test animals and environmental conditions:
no details given, as the primary publication is not available
Type of coverage:
not specified
Preparation of test site:
other: intact or abraded
Vehicle:
unchanged (no vehicle)
Controls:
other: the untreated skin served as control
Amount / concentration applied:
not given
Duration of treatment / exposure:
not given
Observation period:
not given
Number of animals:
not given
Details on study design:
Benzylidene acetone applied full strength to intact or abraded rabbit skin was mildly irritating (Levenstein, 1972b).
Irritation parameter:
other: qualitative result
Remarks on result:
other: mildly irritating
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Remarks on result:
not measured/tested
Irritant / corrosive response data:
Benzylidene acetone applied full strength to intact or abraded rabbit skin was mildly irritating (Levenstein, 1972b).
Interpretation of results:
Category 3 (mild irritant) based on GHS criteria
Conclusions:
Benzylidene acetone applied full strength to intact or abraded rabbit skin was mildly irritating (Levenstein, 1972b).
Executive summary:

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 not irritating in humans (Opdyke, 1973, Fragrance raw materials monographs - benzylidene acetone).

In there it is stated that Benzylidene acetone applied full strength to intact or abraded rabbit skin was mildly irritating (Levenstein, 1972b).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999-07-26 - 1999-07-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Charles River, Deutschland GmbH, Kißlegg
- Weight at study initiation: 2.2 kg / 1.8 kg / 1.9 kg / 1.7 kg
- Housing: individually in PPO cages (floor area: 2576cm²) with perforated floor.
- Diet (e.g. ad libitum): pelleted complete rabbit diet "Altromin 2123" from Altromin, D.32791, Lage, Lippe was available ad libitum (Analyses for major nutritive components and relevant possible contaminants are performed regularly on the diet.)
- Water (e.g. ad libitum): free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth (analyses for relevant possible contaminants are performed regularly).
- Acclimation period: pre-period of at least one week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3 °C
- Humidity (%): 55 +/- 15 %
- Air changes (per hr): 10 / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours darkness

- Other: the animals were earmarked on arrival.
Vehicle:
unchanged (no vehicle)
Controls:
other: the right eye remained untreated and served as control.
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of warmed test article (The test article was warmed up in an incubator to 40 °C and placed there at this temperature for 21 hours. So it turned into a yellowish liquid.)
Duration of treatment / exposure:
24 hours
Observation period (in vivo):
up to 72 h post exposure
Number of animals or in vitro replicates:
4
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): 24 h after instillation of the test substance, eyes were treated with fluorescein and then rinsed with 20 ml 0.9% sodium chloride solution. Thereafter the staining was evaluated using a UV-light. The same procedure was conducted 48 hours and 72 hours after instillation of the test material
- Time after start of exposure: 24 hours

SCORING SYSTEM:
Cornea - Opacity degree of density (Area most dense taken for reading):
0 - No ulceration or opacity
1 - Scattered or diffuse area of opacity (other than slight dulling of normal luster), details of iris clearly visible.
2 - Easi1y discernible translucent areas, details of iris slightly obscured.
3 - Nacreous areas, no details of iris visible, size of pupil barely discernible.
4 - Complete comeal opacity, iris not discernible.

Cornea - Area of cornea involved
1 - One quarter (or less) but not zero.
2 - More than 1 quarter, but less than half.
3 - More than half, but less than 3 quarters.
4 - More than 3 quarters, up to whole area.

Iris
0 - Normal
1 - Markedly deepened folds, congestion, swelling, moderate circumcorneal injection (any of these or combination of any thereof), iris still reacts to light (sluggish reaction is positive).
2 - No reaction to light, haemorrhage, gross destruction (any or all of these).

Conjunctiva - Redness:
0 - Vessels normal
1 - Some vessels definitely injected.
2 - Diffuse, crimson red, individual vessels not easily discernible
3 - Diffuse beefy red

Conjunctiva - Chemosis:
0 - no swelling
1 - any swelling above normal (includes nictating membane
2 - Obvious swelling with partial eversion of lids
3 - Swelling with lids about half closed
4 - Swelling with lids more than half closed.

Conjunctiva - Discharge:
0 - no discharge
1 - Any amount different from normal (does not include small amounts observed in inner canthus of normal animals).
2 - Discharge with moistening of the lids and hairs just adjacent to lids.
3 - Discharge with moistening of the lids and hairs, and considerable area around the eye.

TOOL USED TO ASSESS SCORE: fluorescein

0.1 ml of the test article was placed in the left eye of the rabbits by gently pulling the lower lid away from the eyeball to form a cup into which the article was dropped. The lids were then gently held together for one second. The eyes were examined and the grade of ocular reaction was recorded one hour and 24 hours later. After the first 24 hour reading Fluorescein was instilled. After rinsing with 20 ml 0.9% sodium chloride solution the eyes were examined again using UV-light to detect possible corneal damage. The eyes were also examined 48 and 72 hours after the treatment.
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
other: also 1 h
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
other: also 1h
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 24 h
Remarks on result:
other: 1h and 24 h after application all animals showed some conjunctival vessels definitely injected. Some swelling above normal was seen in all animals after 1 h. After 48 hours as well as after 72 hours all animals were free of any signs of eye irritation.
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 24 h
Remarks on result:
other: after 1 hour slight chemosis was noted in all animals, this however was not evident no more at the 24 h reading.
Irritant / corrosive response data:
The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated:
Cornea opacity: 0.0
Iris lesion: 0.0
Redness of conjunctiva: 0.3
Oedema of conjunctiva: 0.0

Individual scores are found in Table 1.
Other effects:
no other effects reported

Table1:Scores for ocular lesions
Rabbit No./ Weight kg 1h. 24hs. 48hs. 72hs. Individual mean score*
F
1598/ 2.2 Cornea opacity, degree 0 0 0 0 0 0.00
Cornea opacity, area 0 0 0 0 0
Iris 0 0 0 0 0.00
Conjunctiva Redness 1 1 0 0 0.33
Chemosis 1 0 0 0 0.00
Discharge 0 0 0 0  
1599/ 1.8 Cornea opacity, degree 0 0 0 0 0 0.00
Cornea opacity, area 0 0 0 0 0
Iris 0 0 0 0 0.00
Conjunctiva Redness 1 1 0 0 0.33
Chemosis 1 0 0 0 0.00
Discharge 0 0 0 0  
1600/ 1.9 Cornea opacity, degree 0 0 0 0 0 0.00
Cornea opacity, area 0 0 0 0 0
Iris 0 0 0 0 0.00
Conjunctiva Redness 1 1 0 0 0.33
Chemosis 1 0 0 0 0.00
Discharge 0 0 0 0  
1601 / 1.7 Cornea opacity, degree 0 0 0 0 0 0.00
Cornea opacity, area 0 0 0 0 0
Iris 0 0 0 0 0.00
Conjunctiva Redness 1 1 0 0 0.33
Chemosis 1 0 0 0 0.00
Discharge 0 0 0 0  
F Reading after instillation of Fluorescein.
* Only the scores from the readings after 24, 48 and 72 hours are included in the calculation of the individual mean scores.
Interpretation of results:
other: EU GHS criteria not met
Conclusions:
The study was performed according to the OECD TG 405 and EU Method B.5 without deviations and according to the good laboratory practice principles, it is considered to be of the highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. The test material did not induce any irritation or corrosion on rabbit eyes. The test material was considered to be not irritating under the conditions of the test.
Executive summary:

The substance benzalacetone was investigated for its potential for eye irritation in rabbits in 1999 (Schreiter, 1999) according to the method recommended in the OECD Guideline No. 405, "Acute Eye Irritation/Corrosion", Feb. 1987, and EEC Guideline B.5 "Acute Toxicity (Eye Irritation)", Jan. 1997. For the test 0.1 mL of undiluted substance were introduced into the conjunctival sac of the right eye of four rabbits. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing. After a period of 24 h the eye was stained with fluoreszein and carefully washed with 20 mL of physiological sodium chloride solution. The signs of cornea (opacity and area affected), iris (lesion), conjunctivae - (redness, chemosis), and discharge were evaluated as described by DRAIZE. The examinations of cornea, iris and conjunctiva were facilitated by use of optical instruments (e. g. hand slit-lamp). To define epithelial damage, fluorescein was applied to the corneal surface. Very slight signs of irritation were observed on the treated eyes. The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated: Cornea opacity: 0.0, Iris lesion: 0.0, Redness of conjunctiva: 0.3, Oedema of conjunctiva: 0.0.

The results show that the test compound can be regarded as 'not irritating to the eye, as only slight conjunctivae reactions was seen in the animals 1 and 24 h after application of the test substance, which was fully reversible within the observation time. According to the Regulation (EC) No 1272/2008, the test material does not meet the criteria for classification and will not require labelling as irritating to the eyes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

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 sensitisers. Since this test avoids the use of an increase in concentration to maximize the reaction nor does 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.

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 not irritating in humans (Opdyke, 1973, Fragrance raw materials monographs - benzylidene acetone).

In there it is stated that Benzylidene acetone applied full strength to intact or abraded rabbit skin was mildly irritating (Levenstein, 1972b). In there it is also stated that Benzylidene acetone tested in a 2 % concentration in petrolatum produced no irritation in a 48 hr closed-patch test in 25 human subjects (Kligman, 1972). In conclusion, it has to be stated that the test item is irritating to skin, when exposure occurs to higher concentrations of benzalacetone. Therefore the substance had to be classified as a skin irritant.

Eye irritation:

The substance benzalacetone was investigated for its potential for eye irritation in rabbits in 1999 (Schreiter, 1999) to the method recommended in the OECD Guideline No. 405, "Acute Eye Irritation/Corrosion", Feb. 1987, and EEC Guideline B.5 "Acute Toxicity (Eye Irritation)", Jan. 1997. For the test 0.1 mL of undiluted substance were introduced into the conjunctival sac of the right eye of four rabbits. The eyes were examined and the changes were graded according to a numerical scale one hour, 24, 48 and 72 hours after dosing. After a period of 24 h the eye was stained with fluorescein and carefully washed with 20 mL of physiological sodium chloride solution. The signs of cornea (opacity and area affected), iris (lesion), conjunctivae - (redness, chemosis), and discharge were evaluated as described by DRAIZE. The examinations of cornea, iris and conjunctiva were facilitated by use of optical instruments (e. g. hand slit-lamp). To define epithelial damage, fluorescein was applied to the corneal surface. Very slight signs of irritation were observed on the treated eyes. The following mean values, based on the results from the 24, 48 and 72 hour readings, were calculated: Cornea opacity: 0.0, Iris lesion: 0.0, Redness of conjunctiva: 0.3, Oedema of conjunctiva: 0.0.

The results show that the test compound is not irritating to the eye, as only slight conjunctivae reactions were seen in the animals 1 and 24 h after application of the test substance, which were fully reversible within the observation time. According to the European regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification and will not require labelling as irritating to the eyes.


Effects on skin irritation/corrosion: irritating

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 irritating to the skin (Cat. 2, H315).

Eye irritation:

According to the Regulation (EC) No 1272/2008, the test material does not meet the criteria for classification and will not require labelling as irritating to the eyes.