Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jan 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

see above

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

EC Number:

614-257-7

Cas Number:

68071-40-9

Molecular formula:

C18H34O4, C20H38O4 and C22H42O4 (mainly)

IUPAC Name:

Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, closed container, away from sunlight

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Bensheim, 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) at ambient temperature.
- 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

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 etc). 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).
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometry (OD490) (Versamax® Molecular Devices). Prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about 1 h at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (to). 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).

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 - 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:

negative control - mean out of all 3 eyes

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

positive control - mean out of all 3 eyes

Value:

55.67

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 3

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 4

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

negative control - mean out of all 3 eyes

Value:

0.054

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Irritation parameter:

fluorescein leakage

Remarks:

permeability

Run / experiment:

positive control - mean of all 3 eyes

Value:

0.812

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: value is corrected

Results 6

Irritation parameter:

fluorescein leakage

Remarks:

permeability

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 7

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

negative control - mean out of all 3 eyes

Value:

1.15

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 8

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

Run / experiment:

positive control - mean out of all 3 eyes

Value:

67.84

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 9

Irritation parameter:

in vitro irritation score

Remarks:

(IVIS)

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:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: yes

Any other information on results incl. tables

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

	IVIS	Mean IVIS
Negative Control	0.78	1.15
	0.77
	1.90
Test substance	-1.43	0.0
	-0.43
	-0.41
Positive control	70.61	67.84
	66.52
	66.38

Table 2: Opacity and Permeability values

	Difference (t130-to) of Opacity
Parameter	Opacity 1	Opacity 2	Opacity 3	Mean opacity value	Permeability at 490 nm	Mean Permeability at 490 nm
Negative control	0	0	1	0.33	0.052	0.054
					0.051
					0.060
Test substance	-1.33*	-0.33*	-0.33*	-	-0.006*	-
					-0.006*
					-0.005*
Positive control	54.67*	55.67*	56.67*	-	1.063*	-
					0.724*
					0.648*

* = corrected values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The eye irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the Bovine Corneal Opacity and Permeability test method (BCOP) in accordance to OECD 437. The testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was concluded not to eye irritating properties, thus no classification applies.

Executive summary:

The eye irritation potential of Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs was evaluated in vitro in the Bovine Corneal Opacity and Permeability test method (BCOP) in accordance to OECD 437.

Acceptance criteria for negative and positive controls as well as variability between replicate measures was met and the study was therefore considered valid.

The scores for cornes opacity and permeability for the testing material (Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs) was comparable to the negative control group and the In-Vitro Irritancy Score (IVIS) values did not indicate any eye irritative properties. Therefore, it was concluded that Reaction product of 2-Propenoic acid and Oxirane, mono[(C12-16-alkyloxy)methyl] derivs does not to have eye irritating properties.

Thus, no classification applies in acordance to CLP (Regulation (EC) No 1272/2008).