ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

adopted in 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Test material information

Constituent 1

Reference substance name:

ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

EC Number:

500-276-7

EC Name:

ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

Cas Number:

89800-10-2

Molecular formula:

{(C10H16O7).(C3H3O)b.[(C6H10O2)m.(C3H3O)a]} (i.e., UVCB substance)

IUPAC Name:

ε-Caprolactone, oligomers, esters with acrylic acid and 2,2,2',2'-tetrakis (hydroxymethyl)-3,3'-oxydipropan-1-ol

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals: at least 9 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue: The isolated eyes were transported in HBSS (with streptomycin / penicillin).
- Time interval prior to initiating testing: The comea were isolated on the same day after delivery of the eyes and were directly used in the BCOP test.
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: streptomycin / penicillin

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration: undiluted

Duration of treatment / exposure:

10 min at 32 ± 1 °C

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

2 h at 32 ± 1 °C

Number of animals or in vitro replicates:

Number of eyes for the test item/ negative control/ positive control: 3 of each

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: A careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, vascularization). Eyes with defects were discarded. The cornea was carefully removed from the eye using a scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. The cornea were directly used in the BCOP test on the same day.

QUALITY CHECK OF THE ISOLATED CORNEAS: yes, eyes were free of defects

TREATMENT METHOD: corneas were incubated horizontally, in a water-bath

REMOVAL OF TEST SUBSTANCE
After incubation the test item or control item, respectively, was rinsed off from the application side with saline.

- POST-EXPOSURE INCUBATION: The incubation medium consisted of MEM (supplemented with 1.1 g / 500 mL sodium bicarbonate, 5 mL / 500 mL L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin). Immediately before starting the test, MEM was supplemented with 1% fetal calf serum (FCS)). The OECD foresees the use of EMEM which is in composition and osmolarity equivalent to the MEM, thus MEM can be used without restriction.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by the amount of light transmission through the cornea using an opacitometer (OP_KiT opacitometer, Electro Design, 63-Riom France). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t130)
- Corneal permeability: After the final opacity measurement was performed, the complete medium was removed from the anterior compartment and replaced by 1 mL of a 0.4% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C. Complete medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate. Passage of sodium fluorescein dye an thus the optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm. The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value - corrected opacity value mean negative control) + (15 x corrected OD490 value)

Depending on the score obtained, the test item is classified into one of the categories according to OECD 437.

DECISION CRITERIA: The decision criteria as indicated in the TG (OECD 437) was used.

VALIDATION CRITERIA: The test will be acceptable if
- the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
- the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

test item - mean out of all 3 eyes

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 2

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

test item - mean out of all 3 eyes

Value:

0.005

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 3

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

test item - mean out of all 3 eyes

Value:

0.08

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

positive control - mean out of all 3 eyes

Value:

92.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, as the values of opacity and permeability (0 and 0.059, respectively) are lower than the upper limit of the historical values (0.33 and 0.08, respectively)
- Acceptance criteria met for positive control: yes, as the IVIS (92.17) falls within two standard deviations of the current historical mean (88.20 ± 21.48)

Any other information on results incl. tables

Table 1: Opacity and Permeability and In-Vitro Irritancy Score (IVIS) values

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS
		Mean		Mean		Mean
Negative Control	0	0.00	0.055	0.059	0.83	0.88
	0		0.064		0.96
	0		0.057		0.86
Positive Control	78.00*	79.33	0.894*	0.856	91.42	92.17
	75.00*		0.849*		87.74
	85.00*		0.824*		97.37
Test substance	0.00*	0.00	0.004*	0.005	0.07	0.08
	0.00*		0.012*		0.19
	0.00*		-0.002*		-0.03

* = corrected values

Applicant's summary and conclusion

Interpretation of results:

other: CLP/ EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified