Glycerol, ethoxylated

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

24 Mar 2011- 20 Apr 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals No. 437, September 07, 2009 (“Bovine Corneal Opacity and Permeability Test, Method for Identifying Ocular Corrosives and Severe Irritants”)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF SE, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Glycerol, ethoxylated

EC Number:

500-075-4

EC Name:

Glycerol, ethoxylated

Cas Number:

31694-55-0

Molecular formula:

(C2 H4 O)n (C2 H4 O)n (C2 H4 O)n C3 H8 O3 3n =>1-<6.5 mol EO

IUPAC Name:

Alkoxylation reaction product of glycerin as starter and ethylene oxyde (EO) as monomer

Details on test material:

- Name of test material (as cited in study report): Lupranol VP 9209
- Test item number: 11/0080-1
- Physical state: liquid, colorless, clear
- Analytical purity: 100%
- Lot/batch No.: #113
- Expiration date of the lot/batch: 2011-06-13
- Storage condition of test material: room temperature
- pH value (undiluted test substance): 6

Test animals / tissue source

Species:

other: in vitro study

Strain:

other: in vitro study

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- The test system (target tissue): isolated bovine cornea.
Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).
- Supplier: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: highly deionized water

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 μL of test-substance preparation was applied directly to the epithelial surface of the cornea using a syringe (open chamber method)

Duration of treatment / exposure:

10 min

Observation period (in vivo):

Not applicable

Number of animals or in vitro replicates:

Each treatment group (test substance, NC and PC) consisted of 3 corneas

Details on study design:

EXPERIMENTAL PROCEDURE:
-Preparation of the bovine corneas and measurement of initial corneal opacity: corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 566 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.

-Application of the test substance and washing: each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. The undiluted, viscous test substance could not be applied with a pipette. Therefore 750 μL of the test substance was applied directly to the epithelial surface of the cornea using a syringe (open chamber method). Control tissues were concurrently applied into the anterior chamber with 750 μL of highly deionized water (negative control, NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in highly de-ionized water (positive control, PC) using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.
-Post-exposure incubation for liquid test substances and surfactants: the corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.
-Measurement of final corneal opacity: before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.
-Determination of permeability: for determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION:
The mean corneal opacity and permeability values of each treatment group were used to calculate an In Vitro Irritancy Score (IVIS). Calculation of the corneal opacity value: first, the opacity was calculated using the opacitometer specific algorithm.
Then the opacity change per cornea was calculated by subtracting the initial from the final opacity.
• opacity change per cornea = final opacity - initial opacity
Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control.
• corrected opacity change = opacity change - mean opacity change of NC
Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group.
• mean opacity value = mean of all corrected opacity changes per group

-Calculation of permeability value: first, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea.
• OD490 value = OD490 - mean blank OD490
If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value:
• OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490)
Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control.
• corrected OD490 value = OD490 value - mean OD490 value of NC
Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group.
• mean OD490 value = mean of all corrected OD490 values per group

-Calculation of the In Vitro Irritancy Score (IVIS): the IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined:
• IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change
• IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

ACCEPTANCE CRITERIA:
In case one of the below given acceptance criteria is not covered, repetition of the test was considered. A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean. The negative control responses should result in opacity and permeability values that are less than the established upper limits. Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION OF RESULTS:
IVIS >55: risk of serious damage to the eyes
IVIS ≤ 55: no risk of serious damage to the eyes

HISTORICAL CONTROL DATA:
Historical control values of negative and positive controls, gathered over an appropriate time period, were available. These data demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria for the test system.

Results and discussion

In vivo

Irritant / corrosive response data:

Based on the observed results and applying the evaluation criteria, the test substance does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Any other information on results incl. tables

 Table 1: Summary of opacity scores, Permeability scores and in vitro irritancy scores (IVIS)

 

Test item	Cornea number	Opacity score		Permeability score		In vitro irritancy score (IVIS)
		Corrected opacity change	Group mean ± SD	Mean Corrected OD490	Group mean ± SD	Per cornea	Group mean ± SD
Test substance	13	1.6	2.8 ± 1.2	0.021	0.008 ± 0.02	2.0	3.0 ± 1.2
	14	2.9		-0.016		2.7
	15	4.0		0.019		4.3
Negative control substance	1	NA	2.4 ± 3.2	NA	0.016 ± 0.031	5.2	2.6 ± 3.3
	2	NA		NA		-1.1
	3	NA		NA		3.8
Positive control substance	4	115.2	133 ± 15.8	2.479	3.397 ± 0.519	160.0	184 ± 20.9
	5	145.4		3.119		193.8
	6	138.5		2.456		198.2
NA: not applicable; SD: standard deviation

 

Table 2: Historical control values of the negative control (NC) and the positive control (PC). (SD = standard deviation)

	Historical range of NC Historical period: Jan 2010 – Apr 2011
	Mean	SD	Mean+2 SD	Mean–2 SD
Opacity	1.3	1.6	4.5	-2.0
Permeability	0.012	0.019	0.049	-0.025
	Historical range of PC (1% NaOH) Historical period: Jan 2010 – Apr 2011
Opacity	121.3	20.8	163.0	79.6
Permeability	2.340	0.609	3.557	1.123
In vitro irritation score (IVIS)	156.4	24.8	206.0	106.8

 

 

Applicant's summary and conclusion

Interpretation of results:

other: no risk of serious eye damage