Calcium dilactate

Eye irritation

Administrative data

Endpoint:

eye irritation

Remarks:

other: ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

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

Test material information

Constituent 1

Reference substance name:

88% lactic acid

IUPAC Name:

88% lactic acid

Details on test material:

Purac Purac HS88: L+ lactic acid 88% aqueous solution

Test animals / tissue source

Species:

other: Chicken Enucleated Eye Test

Test system

Vehicle:

other: applied neat

Amount / concentration applied:

Concentration: 100 %
Amount applied: 0.03 ml

Duration of treatment / exposure:

10 seconds

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 were obtained from poultry slaughterhouse v.d. Bor, Amersfoortseweg 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 they 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 superfus.ion 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 1 4 4 1 , 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 t'ne cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced, if necessary. Per test sample, three eyes were selected for testing, whereas one additional eye was rinsed with isotonic saline only and served as a control of the experimental conditions. 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. At time t = O, i . e . immediately after the zero reference measurement, each of the three test samples was applied to the designated test eyes. The following procedure applied for each test eye:
The clamp holding the eye was placed on paper tissues outside the chamber with the cornea facing upwards. Each of the two liquid samples was applied in an amount 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 tho- roughly with 20 ml of isotonic saline of ambient temperature. After rinsing, each eye in the holder was returned to its chamber.
The control eye and test eyes were examined at O, 30, 75, 120, 180 and 240 minutes after treatment, using the criteria and scoring system, given in the annex. 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

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 180 and 240 minutes

Score:

3

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: corneal swelling could not be measured at 30, 75 and 120 minutes time interval due to severe to complete corneal opacity

Results 2

Irritation parameter:

other: Opacity

Basis:

mean

Time point:

other: 30, 75, 120, 180, 240 minutes

Score:

4

Max. score:

4

Reversibility:

not specified

Results 3

Irritation parameter:

other: Fluorescein

Basis:

mean

Time point:

other: 30 minutes

Score:

4

Max. score:

4

Reversibility:

not specified

Remarks on result:

other: Fluorescein retention was only scored at 30 minutes after treatment

Irritant / corrosive response data:

After treatment, severe to complete corneal opacity was observed in the three test eyes, which hampered the measurement of corneal thuckness at the 30, 75 and 120 minutes after treatment. At 180 and 240 minutes after treatment, corneal thickness could be measured again and at 240 minutes a maximum mean corneal swelling of 28% was determined. All three eyes showed severe fluorescein retention by damaged epithelial cells. The categories defined for corneal swelling, corneal opacity, and fluorescein retention were: III, IV, and IV.

Applicant's summary and conclusion

Interpretation of results:

highly irritating

Remarks:

Migrated information Criteria used for interpretation of results: not specified

Conclusions:

Classification: the lactic acid sample (HS88) can be considered severely irritating to eyes (R41).

Executive summary:

Lactic acid 88% (sample code HS88), was examined undiluted for eye irritating/corrosive potential in an ex vivo bioassay, namely the Enucleated Eye Test with chicken eyes (CEET). The eyes were collected as waste material from a slaughter-house for chickens, which were killed for human consumption.

HS88 induced severe corneal effects. 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: HS88 can be considered severely irritating to eyes (R41)