Registration Dossier

Administrative data

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:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): benzalacetone
- Substance type: organic
- Physical state: solidified mass, yellowish, solidification point about 38°C - 39°C
- Analytical purity: undiluted, probably techn. pure material was tested
- Stability under test conditions: minimum 1 year
- Storage condition of test material: cool and dark

Test animals / tissue source

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.

Test system

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 h and 72 hours after instillation of the test material
- Time after start of exposure: 24 h

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.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: cornea opacity
Basis:
mean
Time point:
other: mean score - all timepoints (1 h / 24 h / 48 h / 72 h)
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
other: iris lesion
Basis:
mean
Time point:
other: mean score - all timepoints (1 h / 24 h / 48 h / 72 h)
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
other: redness of conjunctiva
Basis:
mean
Time point:
other: mean score - all timepoints (1 h / 24 h / 48 h / 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:
other: oedema of conjunctiva (chemosis)
Basis:
mean
Time point:
other: mean score - all timepoints (1 h / 24 h / 48 h / 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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: EU-GHS
Conclusions:
The study was performed according to the OECD TG405 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/Con-osion", 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 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.