Registration Dossier

Administrative data

Endpoint:
eye irritation
Remarks:
other: Chicken Enucleated Eye Test (validated alternative test)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
- Burton, A.B.G. (1971) A method for the objective assessment of eye irritation. Food and Cosmetics Toxicology 10, 209-217.
- Burton, A.B.G., M. York and R.S. Lawrence (1981) The in vitro assessment of severe irritants. Food and Cosmetics Toxicology 19, 471-480.
- Commission of the European Communities (1991) Collaborative study on the evaluation of alternative methods to the eye irritaüon test. EC Document XI/632/91, V/E/1/131/91.
- Koëter, H.B.W.M. and M.K. Prinsen (1985) Introduction of an in vitro eye irritation test as a possible contribution to the reduction of the number of animals in toxicity testing. TNO Report V85.188/140322, May 1985, Dept. of Biological Toxicology, TNO Toxicology and Nutrition Institute, Zeist, The Netherlands (available upon request).
- Koëter, H.B.W.M. and M.K. Prinsen (1985) Comparison of in vivo and in vitro eye irritancy test systems: A study with 34 substances. Alternative methods in Toxicology, Volume 3, Chapter A9. Mary Ann Liebert, Inc., publishers.
- Price, J.B.and IJ. Andrews (1985) The in vitro assessment of eye irritancy using isolated eyes.
Food and Chemical Toxicology 23 (2)., 313-315. - Prinsen, M.K.and H.B.W.M. Koëter (1985) EC-Validation study on alternatives to the Draize eye irritation test. Pilot Interlaboratory comparison of the enucleated eye test. TNO Report V89.464, April 1990, Dept. of Biological Toxicology, TNO Toxicology and Nutrition Institute, Zeist, The Netherlands (available upon request).
- Prinsen, M.K. and H.B.W.M. Koëter (1994) Justification of the Enucleated Eye Test with eyes of slaughterhouse animals as an alternative to the Draize eye irritation test with rabbits. Food and Chemical Toxicology, Volume 31, l, 69-76.
- Prinsen, M .K . The Chicken Enucleated Eye Test (CEET): a practical (pre)screen for the assessment of eye irritation/corrosion potential of test materials. Food and Chemical Toxicology, scheduled to be published in Volume 34, no. 4, April 1996.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
other: Chicken
Strain:
other: ROSS, spring chickens
Details on test animals or tissues and environmental conditions:
Approximately 7 weeks old, male or female chickens (Ross, spring chickens), body weight range approximately 2.5 - 3.0 kg, were used as eye-donors.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: control eye
Amount / concentration applied:
0.03 ml
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
4 hours
Number of animals or in vitro replicates:
4 eyes
Details on study design:
Approximately 7 weeks old, male or female chickens (Ross, spring chickens), body weight range approximately 2.5 - 3.0 kg, were used as eye-donors. Heads of these animals wer obtained from poultry slaughterhouse v.d. Bor, Amerfoortseweg 118, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incission of the neck for bleeding, and before thery reached the next station on the process line. The heads were placed in small plastic boxes (3 heads per box) on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature. Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2% w/v (Minims, Smith & Nephew Ltd., Romford, England) was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline of ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 CN, Haag-Streit AG, Liebefeld-Bern, Switzerland), to ensure that the cornea was not damaged. If undamaged, the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of ca 0.10 - 0.15 ml/min (peristaltic pump, Desaga STA 131900, Heidelberg, Germany). The chambers of the superfusion apparatus as well as the saline were temperature controlled at 32 ± 1.5 °C (waterpump, Thermomix 1441 , B. Braun Melsungen AG, Melsungen, Germany). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. Corneal thickness was measured using the Depth Measuring Attachment no. II for the Haag-Streit slit-lamp microscope. Thickness of the cornea was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, or eyes that were unacceptably stained with fluores- cein (score higher than 0.5), indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced, if necessary. Four eyes were selected for testing. After an equilibration period of 45-60 minutes, the corneal thickness of the eyes were measured again to determine the zero reference value for corneal swelling calculations. The sample of the test substance was tested undiluted on three test eyes and one additional eye was rinsed with isotonic saline only and served as a control of the experimental conditions. At time t = 0, i.e . immediately after the zero reference measurement, the test substance was applied to the eye. For this purpose, the clamp holding the eye was placed on paper tissues outside the chamber with the cornea facing upwards. The test substances was applied in amounts of 0.03 ml from a micropipette (Nichiryo Co., Ltd., model 8100, Tokyo, Japan), in such a way that the entire surface of the cornea was bathed with the test material. With the solid sample, the cornea was powdered with 0.03 g of the test material. After a total exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 ml of isotonic saline of ambient temperature. After rinsing, each eye in the holder was returned to its chamber. This procedure was repeated for each test eye.
The control eye and test eyes were examined at 0, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system, given in the appendix. Fluorescein retention was only scored at 30 minutes after treatment. All examinations were carried out with the slit-lamp microscope.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
other: corneal swelling
Basis:
other: Enucleated eye
Time point:
other: 240
Score:
3
Reversibility:
other: not relevant
Irritation parameter:
other: corneal opacity
Basis:
other: Enucleated eye
Time point:
other: 240
Score:
3
Reversibility:
other: not relevant
Irritation parameter:
other: fluorescein retention
Basis:
other: Enucleated eye
Time point:
other: 240
Score:
4
Reversibility:
other: not relevant
Irritant / corrosive response data:
After treatment, the thickness of the cornea of the test eyes gradually increased considerably; a maximum mean corneal swelling of 31% was obtained at 240 min after treatment. Moderate or severe corneal opacity and severe fluorescein retention by damaged epithelial cells were observed in the test eyes. In addition, the three test eyes showed wrinkling of the corneal epithelium. The irritancy categories assigned to these findings for corneal swelling, corneal opacity, and fluorescein retention were: III, III and IV.

Applicant's summary and conclusion

Interpretation of results:
moderately irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Classification: ethyl lactate can be considered severely irritating to eyes (R41).
Executive summary:

In an ex vivo bio assay, the Chicken Enucleated Eye Test (CEET), 0.03 ml of undiluted ethyl lactate was applied to enucleated chicken eyes for 10 seconds, after which the corneal surface was rinsed with 20 ml of isotonic saline. The eyes were then examined up to 4 hours after treatment. Irritation was scored as thickness of the cornea, corneal opacity and fluorecein retention.

After treatment, the thickness of the cornea of the test eyes gradually increased considerably; a maximum mean corneal swelling of 31% was obtained at 240 min after treatment. Moderate or severe corneal opacity and severe fluorescein retention by damaged epithelila cells were observed in the test eyes. In addition, the three test eyes showed wrinkling of the corneal epithelium. On the basis of the results obtained with this in vitro (ex vivo) assay and according to the scheme for (EC-)classification applied, the following was concluded: ethyl lactate is moderately irritating to eyes, but in view of the additional effects observed, it is recommended to classify ethyl lactate as R41: risk of serious damage to eyes.