Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1994–September 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Principles of method if other than guideline:

The test substance was also examined in an alternative ex vivo bioassay, namely the Enucleated Eye Test with chicken eyes (CEET).

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

EC Number:

606-097-1

Cas Number:

186817-80-1

Molecular formula:

C11H22O3

IUPAC Name:

Propanoic acid, 2-hydroxy-, 2-ethylhexyl ester, (2S)-

Constituent 2

Reference substance name:

2-ethylhexyl-S-lactate

IUPAC Name:

2-ethylhexyl-S-lactate

Details on test material:

Trade name: Purasolv EHL
Purity: min. 97 %

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Broekman Instituut B.V., Someren, the Netherlands
- Age at study initiation: young adult
- Weight at study initiation: 2250-2940 g
- Housing: individually in a stainless steel cage, fitted with a perforated floor
- Diet (e.g. ad libitum): standard laboratory rabbit diet, ad libitum
- Water (e.g. ad libitum):tap water, ad libitum
- Acclimation period: 27 or 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 42-82.5 % (upper limit higher than the intended 70 %, because of meteorological circumstances or because of wet cleaning of the animal room; the 82.5 % peak occurred for ca one hour at most)
- Air changes (per hr): ca 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours dark/ 12 hours light

IN-LIFE DATES: May 18, 1994 or June 1, 1994 to July 8, 1994

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 ml 2-ethylhexyl lactate

Duration of treatment / exposure:

Single application

Observation period (in vivo):

25 days

Number of animals or in vitro replicates:

3

Details on study design:

In vivo test:
An amount of 0.1 ml of the test substance was instilled in the conjunctival dul-de-sac of the right eye. After administration, the upper and lower eye lid were carefully closed and subsequently held together for at least one second before releasing, to prevent loss of material. The left eye remaining untreated, served as a control.
The reactions of the test eyes were judged at circa one hour, 24, 48, and 72 hours, and at 7, 14, 21 and 25 days after treatment using the scoring scale which is given in the appendix in the section "Attached background material".

Ex vivo pre-screening, CEET:
Experimental 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 the 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 in 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 fluorescien-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 postitioned 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). Four eyes were selected and 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 Attachement 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 were replaced. 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 test substance was tested undiluted on three test eyes. At time t = 0, i.e. immediately after the zero reference measurement, the appropriate sample was applied to the cornea. For this purpose, the clamp holding the eye was placed on a paper tissue outside the chamber with the cornea facing upwards. The liquid material was applied in amounts of 0.03 ml from a micropipette (Nichirryo Co., Ltd., model 8100, Tokyo, Japan), in such a way that the entire surface of the cornea was batched with the test substance. After a total exposure period of 10 seconds, the corneal surface was rinsed thoroughly with 20 ml of isotonic saline of ambient temperature. Next, the eye in the holder was returned to its chamber. This procedure was repeated each test eye. The control was treated with saline only. The eyes were examined at 30, 75, 120, 180, and 240 minutes after treatment, using the criteria and scoring system, given in the appendix 2 in the section "Attached background material". All examinations were carried out with the slit-lamp microscope.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

>= 1 - <= 2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Time point:

24/48/72 h

Score:

>= 1 - <= 2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

> 0 - <= 1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Time point:

24/48/72 h

Score:

>= 0 - <= 1

Max. score:

2

Reversibility:

fully reversible within: 7 days

Results 7

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

>= 2 - <= 3

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 8

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #2

Time point:

24/48/72 h

Score:

>= 3

Max. score:

3

Reversibility:

fully reversible within: 21 days

Results 9

Irritation parameter:

conjunctivae score

Remarks:

redness

Basis:

animal #3

Time point:

24/48/72 h

Score:

>= 1 - <= 2

Max. score:

3

Reversibility:

fully reversible within: 7 days

Results 10

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

ca. 1

Max. score:

4

Reversibility:

fully reversible within: 14 days

Results 11

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

>= 1 - <= 2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Results 12

Irritation parameter:

chemosis score

Basis:

animal #3

Time point:

24/48/72 h

Score:

>= 1

Max. score:

4

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

At 1 hour after treatment, the eye effects observed in the three rabbits consisted of slight corneal opacity, slight iritis, slight redness and moderate swelling of the conjunctivae, and severe ocular discharge.
At 24 hours after treatment, the eye effects observed consisted of slight or moderate corneal opacity, slight iritis, moderate or severe redness and slight swelling of the conjunctivae, and slight or severe ocular discharge.
At 48 hours after treatment, the eye effects observed consisted of slight or moderate corneal opacity, slight iritis (two rabbits), moderate or severe redness and slight or moderate swelling of the conjunctivae, and slight or severe ocular discharge.
At 72 hours after treatment, the eye effects observed consisted of slight or moderate corneal opacity, slight iritis (one rabbit), slight, moderate or severe redness and slight or moderate swelling of the conjunctivae, and moderate ocular discharge (one rabbit).
At 7 days after treatment, the eye effects observed consisted of slight corneal opacity and vascularization of the cornea in one rabbit, and slight redness and slight swelling of the conjunctuvae in two rabbtis.
At 14 days after treatment, eye effects were still observed in one rabbit and consisted of slight corneal opacity, vascularization of the cornea, and slight redness and slight swelling of the conjunctivae.
At 21 days, these eye effects except minor vascularization of the cornea had cleared completely.
At 25 days, this eye effect had also cleared completely.

Other effects:

Vascularization of the cornea occurred in one rabbit at day 14. The degree diminished gradually,and had disappeared completely at day 25.
Results for the ex vivo study according to CEET:
Mean values for corneal swelling, corneal opacity, and fluorescein retention are presented in Table 2.
After treatment, the thickness of the cornea of the test eyes gradually increased; a maximum mean corneal swelling of 18 % was obtained at 240 min after treatment. Moderate corneal opacity and moderate fluorescein retention by damaged epithelial cells were observed in the three test eyes.
The irritancy categories assigned to these findings are also presented in Table 2, together with the final irritancy classification. The categories defined for corneal swelling, corneal opacity, and fluorescein retention were: II, III, and III.

Any other information on results incl. tables

Table 1. Individual scores awarded to the ocular lesions elicited by 2-ethylhexyl-lactate.

rabbit	corneal	iris	conjunctivae		ocular
number	opacity	effects	redness	chemosis	discharge
after one hour
37	1 (4)	1	1	2	3
29	1 (4)	1	1	2	3
30	1 (4)	1	1	2	3
after 24 hours
37	2 (4)	1	3	1	3
29	1 (4)	1	3	1	3
30	2 (4)	1	2	1	1
after 48 hours
37	2 (2)	1	3	1	1
29	2 (2)	1	3	2	3
30	1 (2)	0	2	1	1
after 72 hours
37	2 (2)	0	2	1	0
29	2 (4)	1	3	2	2
30	1 (1)	0	1	1	0
after 7 days
37	0	0	1	1	0
29	1 (4)^1	0	1	1	0
30	0	0	0	0	0
after 14 days
37/30	0	0	0	0	0
29	1 (3)^1	0	1	1	0
after 21 days
29	0^1	0	0	0	0
after 25 days
29	0	0	0	0	0

The scores are explained in the appendix in the attached background material section

() = area of opacity; 1 = one quarter (or less) but no zero, 2 = half area, 3 = three quarters, 4 = whole area.

¹= vascularization of the cornea; the degree diminished gradually

Table 2. Mean values for corneal swelling, corneal opacity and fluorescein retention values of the test eyes treated with 2-ethylhexyl-lactate, and the irritancy categories based on the maximum mean scores, and the final EC-classification

Time intervals	Corneal swelling %	Corneal opacity	Fluorescein retention
30	7 (1)	1.0 (0)	2.0 (0.0)
75	9 (2)	1.5 (0)
120	14 (3)	2.0 (0)
180	16 (4)	2.0 (0)
240	18 (5)	2.0 (0)

 

Parameter	Maximum score	Category
Corneal swelling	18	II
Corneal opacity	2	III
Fluorescein retention	2	III
EC-classification: Irritating to eyes (R36)

between brackets = mean standard error of the mean

Applicant's summary and conclusion

Interpretation of results:

irritating

Conclusions:

2-Ethylhexyl-lactate is distinctly irritating for the eyes of rabbits, and needs labelling as "Category 2: irritating to eyes" according to the EC standards (former label R36, irritating to eyes).
The results of the alternative test method with enucleated chicken eyes were in agreement with the results of the in vivo rabbit eye test.

Executive summary:

2-Ethylhexyl-lactate caused moderate reaction in CEET. Therefore it was decided to preceed with the in vivo rabbit eye test. In a primary eye irritation study, 0.1 ml of undiluted 2-ethylhexyl-lactate was instilled into the conjunctival sac of the right eye of three young adult New Zealand White rabbits of unknown sex. The eyes were not washed. Animals were observed for 25 days. Irritation was scored by the method of Draize.

The test substance caused moderate corneal opacity, slight iritis, moderate or severe redness and moderate swelling of the conjunctivae and severe ocular discharge in the three rabbits. In addition, one rabbit showed vascularization of the cornea. At 21 days all effects except the vascularization in the one rabbit had cleared; at day 25 this effect had also cleared completely.

In this study, 2-ethylhexyl-lactate is an eye irritant based on EC standards and needs labeling as "Category 2, irritating to eyes" (formerly R36).