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Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 April 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

Constituent 1
Chemical structure
Reference substance name:
Ethyl 4-methyl-2-oxocyclohexanecarboxylate
EC Number:
236-910-1
EC Name:
Ethyl 4-methyl-2-oxocyclohexanecarboxylate
Cas Number:
13537-82-1
Molecular formula:
C10H16O3
IUPAC Name:
ethyl 4-methyl-2-oxocyclohexane-1-carboxylate
Test material form:
liquid
Details on test material:
Physical appearance: Clear colourless liquid
Storage conditions: In refrigerator (2-8°C) in clear glass bottle
Specific details on test material used for the study:
Stability at higher temperatures: maximum temperature: 50°C, maximum duration: 1440 minutes

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: 750 µL

NEGATIVE CONTROL: Physiological saline
- Amount applied: 750 µL

POSITIVE CONTROL: Ethanol
- Amount applied: 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
Details on study design:
TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µl of either the negative control, the positive control or the test item was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°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.

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score 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 upper limits of the laboratory historical range

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
Mean of 3 replicates
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- The corneas treated with the test item showed opacity values between -1.4 and 0.6, permeability values were ranging from 0.001 to 0.0.26 and IVIS were -1.0, 0.4 and 0.7 (n=3).
- Since the mean IVIS was < 3 after 10 minutes of treatment, the test item is not classified according to GHS and Regulation (EC) No. 1272/2008.

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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within the historical range (IVIS ranging from -0.4 to 0.5)
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 38-59). Corneas were turbid after 10 minutes of treatment.

Any other information on results incl. tables

Table 2 Historical data for the BCOP studies

 

Negative control

Positive control

 

Opacity

Permeability

In vitroIrritancy Score

In vitro Irritancy Score

Range

-2.9 – 3.0

-0.016 – 0.042

-2.8 – 3.0

34.7 – 78.2

Mean

0.12

0.01

0.32

56.68

SD

1.07

0.01

1.17

12.64

n

72

65

66

47

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 2014 to February 2017.

Table 3 Individual permeability scores (corrected)

Treatment

Dilution factor

Negative control corrected OD49011

Negative control corrected OD49021

Negative control corrected OD49031

Negative control corrected OD490 Average

Negative control corrected final OD490

Average OD

 

Positive control

6

0.352

0.354

0.350

0.352

2.112

1.992

1

0.989

0.981

0.984

0.985

0.985

6

0.484

0.475

0.480

0.480

2.878

 

Test item

1

0.025

0.026

0.026

0.026

0.026

 

0.011

 

1

-0.001

0.005

0.000

0.001

0.001

1

-0.002

0.008

0.008

0.005

0.005

1    OD490values corrected for the mean final negative control permeability (0.005).

2    Calculations are made without rounding off.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008
Conclusions:
In the Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, FRET 13-0545 induced a mean IVIS of 0.0 after 10 minutes of treatment. Based on these results, the substance is not classified according to GHS and Regulation (EC) No. 1272/2008.