2,5-furandione, dihydro-, mono- C18- alkenyl derivs. reaction products with potassium hydroxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2017 - 28 February 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:

26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Potassium hydrogen 2-octadecenylsuccinate

EC Number:

296-086-4

EC Name:

Potassium hydrogen 2-octadecenylsuccinate

Cas Number:

92218-39-8

Molecular formula:

C22H40O4.K

IUPAC Name:

hydrogen potassium 2-octadec-1-en-1-ylsuccinate

Test material form:

solid

Details on test material:

Name as cited in report: X-19924
Appearance: thick amber paste
Storage: at room temperature

Specific details on test material used for the study:

- Correction factor for purity: 1.138; this correction factor was not applied in this study since a correction factor is not applicable to the BCOP test.
- Stability at higher temperatures: stable for a maximum of 30 minutes at a maximum temperature of 60ºC

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse Vitelco, 's-Hertogenbosch, The Netherlands.
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected an transported in physiological saline in a suitable container under cooled conditions.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: excessive amount to cover the complete cornea

NEGATIVE CONTROL: 750 µL

POSITIVE CONTROL: 750 µL

Duration of treatment / exposure:

10 ± 1 minutes

Duration of post- treatment incubation (in vitro):

120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

Number of animals or in vitro replicates:

3 for the negative control, the positive control and the treatment group each
Since the first experiment was inconclusive, a second experiment was performed.

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: The eyes were checked for unacceptable defects and those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 °C ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 °C ± 1 °C

TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or an excessive amount of test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 25.0 °C. In the repeat of the second experiment, corneas treated with negative and positive control were incubated in a horizontal position for 10 ± 1 minutes at 32.0 °C ± 1 °C and the test item was incubated in a horizontal position for 10 ± 1 minutes at 28.5°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.

- POST-EXPOSURE INCUBATION: 120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA (see table 1):
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

First experiment; mean of 3 replicates

Value:

3.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS: 0.0 - 1.9

Positive controls validity:

valid

Remarks:

IVIS: 34.0 - 62.2

Remarks on result:

other: results were spread over 2 categories

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Second experiment; mean of 3 replicates

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

IVIS: -1.2 - -0.5

Positive controls validity:

valid

Remarks:

IVIS: 48.2-82.1

Remarks on result:

other: results were spread over 2 categories

Other effects / acceptance of results:

FIRST EXPERIMENT:
- The corneas treated with the test substance showed opacity values between 1.3 and 3.8.
- Permeability values were ranging from 0.023 to 0.060.
- IVIS were 1.8, 3.0 and 4.8 (n=3).

Since the results of the first experiment were spread over 2 categories, the test was inconclussive and a second experiment was performed.

SECOND EXPERIMENT:
- The corneas treated with the test substance showed opacity values between 0.2 and 4.4.
- Permeability values were ranging from 0.003 to 0.040.
- IVIS were 0.9, 2.1 and 5.0 (n=3).

Since the individual in vitro irritancy scores were spread over 2 categories, the test was inconclusive.

OTHER EFFECTS:
- No pH effect of the test item was observed on the rinsing medium in both experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from 0.0 to 1.9 in the first experiment and ranging from 1.2 to -0.5 in the second experiment). However, in the first experiment, one of the negative control eyes was excluded from the analysis since the IVIS was >3. Since the other 2 eyes completely met the criteria and test item results were not influenced by this result this does not affect the study outcome.
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 34 to 62 in the first experiment and ranging from 48 to 82 in the second experiment). In both experiments, the corneas treated with the positive control item were turbid after the 10 minutes of treatment.

Any other information on results incl. tables

Table 2 Historical Control Data for the BCOP Studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9 – 3.0	-0.016 – 0.042	-2.8 – 3.0	35.8-77.0
Mean	0.20	0.01	0.32	57.55
SD	1.02	0.01	1.10	12.12
n	63	59	57	38

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of February 2015 to November 2016.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, no conclusion can be drawn for the classification of X-19924 for eye irritation. The IVIS were spread over 2 categories in a first and in a second experiment (1.8, 3.0, 4.8 and 0.9, 2.1, 5.0, respectively).

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.