Methyl (S)-(-)-lactate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995-06-19 to 1995-07-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl (S)-2-hydroxypropionate

EC Number:

211-694-1

EC Name:

Ethyl (S)-2-hydroxypropionate

Cas Number:

687-47-8

Molecular formula:

C5H10O3

IUPAC Name:

ethyl 2-hydroxypropanoate

Specific details on test material used for the study:

- Name of test material used in the report: ethyl lactate
- CAS number: 687-47-8
- Batch number: 950614
- Appearance: liquid
- Purity: 98 %
- Storage: ambient

Test animals / tissue source

Species:

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:

yes, concurrent no treatment

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 were 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 incision 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 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 (water pump, 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 fluorescein (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 vitro

Results

Results 1

Irritation parameter:

corneal swelling 

Run / experiment:

at 30 min

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

at 30 min

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

at 30 min

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Remarks on result:

other:

Remarks:

fluorescein retention was only determined at t = 30 min

Results 4

Irritation parameter:

corneal swelling 

Run / experiment:

at 75 min

Value:

15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 5

Irritation parameter:

cornea opacity score

Run / experiment:

at 75 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 6

Irritation parameter:

corneal swelling 

Run / experiment:

at 120 min

Value:

24

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 7

Irritation parameter:

cornea opacity score

Run / experiment:

at 120 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 8

Irritation parameter:

corneal swelling 

Run / experiment:

at 180 min

Value:

27

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 9

Irritation parameter:

cornea opacity score

Run / experiment:

at 180 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 10

Irritation parameter:

corneal swelling 

Run / experiment:

at 240 min

Value:

31

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Results 11

Irritation parameter:

cornea opacity score

Run / experiment:

at 240 min

Value:

2.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Other effects / acceptance of results:

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:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Under the given conditions, the test item is considered severely irritating to eyes.

Executive summary:

In an ex vivo bioassay, the Chicken Enucleated Eye Test (CEET), 0.03 mL of undiluted ethyl (S)-lactate (purity: 98 %) 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 fluorescein 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 epithelial cells were observed in the test eyes. In addition, the three test eyes showed wrinkling of the corneal epithelium. Based on this results the test item was considered severely irritating to the eyes. Therefore, classification of ethyl-(S)-lactate as Eye Dam. 1 (H318) according to the CLP Regulation 1272/2008 is warranted.