2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Aug 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted in accordance with internationally recognised test methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate

EC Number:

203-846-0

EC Name:

2,2'-[ethane-1,2-diylbis(oxy)]bisethyl diacetate

Cas Number:

111-21-7

Molecular formula:

C10H18O6

IUPAC Name:

2-[2-(2-acetyloxyethoxy)ethoxy]ethyl acetate

Details on test material:

- Name of test material (as cited in study report): Triethylene Glycol Diacetate [2,2’-{ethane-1,2-diyibis(oxy)}bisethyl Diacetate] CAS No. 111-21-7
- Physical state: clear liquid
- Analytical purity: 99.2%
- Batch number: 57325
- Expiry date: 2015-02-24
- Storage conditions: room temperature, in the dark

Test animals / tissue source

Species:

other: in vitro test with bovine eyes

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

TEST MATERIAL SOURCE AND TREATMENT
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The corneas were prepared immediately on arrival.

PREPARATION OF CORNEAS
The cornea from each selected eye was removed leaving a 2-3 mm rim of sclera to facilitate handling. The iris and lens were peeledaway from the cornea. The isolated corneas were immersed in HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete minimum essential medium (MEM) and plugged. The holders were incubated at 32 ±1ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects and only corneas free of damage were used.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: yes, positive and negative control items were included

Amount / concentration applied:

TEST MATERIAL AND CONTROL SUBSTANCES
- Amount(s) applied: 0.75 mL

Duration of treatment / exposure:

10 min (exposure with the test material)
120 ± 10 min (incubation in MEM)

Observation period (in vivo):

120 ± 10 min after exposure

Number of animals or in vitro replicates:

3 test corneas
3 negative control corneas
3 positive control corneas

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.
- Time after start of exposure: 120 ± 10 min

SCORING SYSTEM: In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

TOOL USED TO ASSESS SCORE: fluorescein

POSITIVE CONTROL SUBSTANCE: ethanol

NEGATIVE CONTROL SUBSTANCE: 0.9% (w/v) sodium chloride solution

PERMEABILITY DETERMINATION: 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured. The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD 492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

OPACITY MEASUREMENT: The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading (corrected for the average change in opacity observed for the negative control corneas). The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

HISTOPATHOLOGY: The corneas were retained after testing for possible conduct of histopathology (in 10% neutral buffered formalin).

VISUAL OBSERVATION: The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

other: Negative Control

Time point:

other: 120 min

Score:

3.5

Results 2

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

other: Test item

Time point:

other: 120 min

Score:

0.4

Results 3

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

other: Positive Control

Time point:

other: 120 min

Score:

37.6

Irritant / corrosive response data:

The corneas treated with the test item were clear post treatment and post incubation.
The corneas treated with the negative control item were clear post treatment and post incubation.
The corneas treated with the positive control item were cloudy post treatment and post incubation.

Any other information on results incl. tables

Table 1. Individual and Mean Corneal Opacity and Permeability Measurements.

Treatment	Cornea number	Opacity					Permeability		In vitro Irritancy score
		Pre-T	Post-T	Post-I	Post-I – Pre-T	Corrected value		Corrected value
Negative control	13	1	3	5	4	-	0160	-	-
	14	1	2	2	1	-	0.176	-	-
	15	1	1	1	0	-	0.035	-	-
	-	-	-	-	1.7*	-	0.124#	-	3.5
Positive control	10	0	18	20	20	18.3	1.290	1.166	-
	11	2	34	35	33	31.3	1.053	0.929	-
	12	0	22	24	24	22.3	0.747	0.623	-
	-	-	-	-	-	24.0**	-	0.906**	37.6
Test item	16	1	4	2	1	0.0	0.028	0.000	-
	17	1	4	2	1	0.0	0.027	0.000	-
	18	3	7	6	3	1.3	0.043	0.000	-
	-	-	-	-	-	0.4**	-	0.000**	0.4

Pre-T: Pre-treatment, Post-T: Post-treatment, Post-I: Post-incubation

*: Mean of the post incubation – pre-treatment values

# = Mean permeability

** = Mean corrected value

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Executive summary:

Potential eye irritation/corrosion has been investigated in an in-vitro BCOP assay using OECD test methods. In accordance with the decision criteria for classification and labelling, as given in OECD guideline 437 (adopted 26 July 2013), the results indicate that the substance does not need to be classified.