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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2018 - 15 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
09 October 2017
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-6-propyl-2H-pyran-2-one
EC Number:
211-820-5
EC Name:
Tetrahydro-6-propyl-2H-pyran-2-one
Cas Number:
698-76-0
Molecular formula:
C8H14O2
IUPAC Name:
6-propyloxan-2-one
Test material form:
liquid
Details on test material:
Appearance: Clear colourless to pale yellow liquid
Storage: In refrigerator (2-8°C)
Specific details on test material used for the study:
No purity correction factor was used.

Test animals / tissue source

Species:
human
Details on test animals or tissues and environmental conditions:
TEST SYSTEM:
The EpiOcular tissue construct is a nonkeratinized epithelium prepared from normal human keratinocytes. It models the cornea epithelium with progressively stratified, but not cornified cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo.
- Justification: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 μL
- The test material was directly applied to the tissues which were pre-wetted with 20 μL of Ca2+Mg2+-Free-DPBS.

POSITIVE CONTROL
- Amount applied: 50 μL Methyl Acetate

NEGATIVE CONTROL
- Amount applied: 50 μL Milli-Q water
Duration of treatment / exposure:
30 minutes ± 2 minutes
Duration of post- treatment incubation (in vitro):
Post-soak: 12 ± 2 minutes; incubation after the post-soak: 120 minutes ± 15 minutes
Number of animals or in vitro replicates:
2 tissues per test item and 2 tissues for the negative and the positive control each.
Details on study design:
TEST PROCEDURE
- RhCE tissue construct used, including batch number: EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27433)
- Duration and temperature: before the assay was started the tissues were incubated at standard culture conditions for 30 ± 2 minutes. The tissues were exposed to the test item for 30 minutes ± 2 minutes at 37.0 ± 1.0°C. After the exposure period, the tissues were thoroughly rinsed with Ca2+Mg2+-free DPBS (brought to room temperature) to remove residual test item.The post-soak immersion was 12 ± 2 minutes at room temperature in assay medium followed by an incubation of 120 minutes ± 15 minutes at 37°C in assay medium. After incubation the cell cultures were exposed to MTT for 180 ± 10 minutes at 37°C.
- The test item was checked for possible color interference by a 1-hour incubation with sterile Milli-Q water and a 2-3 hour incubation with isopropanol, before the study started. Possible direct MTT reduction was checked by a 3-hour incubation with MTT before the study was started.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature: 37.0 ± 1.0°C (actual: 35.8 - 37.2°C)
- Humidity: 80-100% (actual: 47-83%), containing 5.0 ± 0.5% CO2

TISSUE VIABILITY MEASUREMENTS
After the exposure and post-soak periods the tissues were incubated with 0.3 mL MTT-medium (1.0 mg/ml) for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were transferred to a 24-well plate containing 2 mL isopropanol. Formazan was extracted with 2 mL isopropanol refrigerated for 18 ± 2 hours in the dark. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

EVALUATION CRITERIA
- The test chemical is identified as not requiring classification and labelling according to UN GHS (No Category) if the mean percent tissue viability after exposure and post-exposure incubation is more than (>) 60%. In this case no further testing in other test methods is required.
- The test chemical is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-exposure incubation is less than or equal (≤) to 60%.

ACCEPTABILITY CRITERIA
- The absolute mean OD570 of the two tissues of the negative control should reasonably be > 0.8 and < 2.5.
- The mean relative tissue viability of the positive control should be <50% relative to the negative control.
- The difference between the % tissue viabilities of the two identically treated replicates should be <20%.

Results and discussion

In vitro

Results
Irritation parameter:
other: % tissue viability
Run / experiment:
mean of 2 replicates
Value:
5.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no
- Interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: see 'any other information on results' for historical data.
Results for the positive control were within the historical data range and therefore showing that the test system was functioning properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the two tissues of the negative control was between >0.8 and <2.5 (i.e., 1.702)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 13%).
- The difference between the % tissue viabilities of the two identically treated replicates was <8% (i.e., 1.5%, 1.5% and 8% for the negative control, the treatment group and the positive control respectively)

Any other information on results incl. tables

Table 1 Individual OD Measurements at 570 nm

 

A

(OD570)

B

(OD570)

Negative control

OD570 measurement 1

OD570 measurement 2

 

1.7810

1.7333

 

1.7445

1.7186

The test item

OD570 measurement 1

OD570 measurement 2

 

0.1509

0.1503

 

0.1248

0.1254

Positive control

OD570 measurement 1

OD570 measurement 2

 

0.1876

0.1887

 

0.3251

0.3224

OD = Optical density

Duplicate exposures are indicated by A and B.

Table 2 Historical data

 

Negative control

(absorption; OD570)

Positive control

(absorption; OD570)

Positive control

(viability; %)

Range

1.077 – 1.805

0.029 – 0.793

2.11 – 48.25

Mean

1.52

0.42

26.86

SD

0.21

0.23

14.11

n

16

16

16

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 January 2013 to May 2017.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
Conclusions:
Based on the results of an in vitro skin corrosion EpiOcular test, Tetrahydro-6propyl-2H-pyran-2-one (delta octalactone) is potentially irritant or corrosive to the eyes with a mean cell viability of 5.6% compared to the negative control. Therefore, the test item potentially requires classification and labelling according to UN GHS (Category 2 or Category 1).