γ-valerolactone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-07-13 to 2020-11-17

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: B.69 Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage; Official Journal of the European Union, No.L 247

Version / remarks:

31 July 2019

Deviations:

no

Test guideline 2

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:

18 June 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

γ-valerolactone

EC Number:

203-569-5

EC Name:

γ-valerolactone

Cas Number:

108-29-2

Molecular formula:

C5H8O2

IUPAC Name:

5-methyloxolan-2-one

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch number of test material: Grosss 542
- Expiration date of the batch: 04 June 2022

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

Test animals / tissue source

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: OECD accepted in vitro model as part of a turnkey test strategy to assess the eye irritation potential of chemicals
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The EpiOcularTM model (OCL-200) is a three-dimensional, non-keratinized tissue construct composed of normal human-derived epidermal keratinocytes used to model the human corneal epithelium. The EpiOcular tissues (surface 0.6 cm²) are cultured on cell culture inserts (MILLICELLs, 10 mm ∅) and are available commercially as kits (EpiOcular™ 200) containing 24 tissues on shipping agarose. The cells used to produce EpiOcular tissue are screened for potential biological contaminants. No contaminations have been detected in this tissue batch.
- RhCE tissue used, including batch number: All cells used to produce EpiOcular are purchased or derived from tissue obtained by MatTek Corporation from accredited institutions (keratinocyte strain 4F1188, Lot No 30669)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 μL

Duration of treatment / exposure:

30 minutes

Duration of post- treatment incubation (in vitro):

After 12 minutes of post-soak immersion, the tissues were incubated at standard culture conditions for 2 hours (post-incubation period)

Number of animals or in vitro replicates:

a single test composed of 2 tissue replicates

Details on study design:

- Details of the test procedure used: Using a pipette, fifty microliters (50 μL) undiluted liquid test substance were applied covering the whole tissue surface. Control tissues were applied concurrently with 50 μL sterile deionized water (NC) or with 50 μL methyl acetate (PC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.
- Doses of test chemical and control substances used: 50 µL of test chemical/positive control/negative control
- Duration and temperature of exposure and post-exposure incubation periods: Exposure at 37°C for 30 minutes, then post-exposure incubation for 2 hours at 37°C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: Prior to testing, the test substance was added to 0.9 mL MTT solution. The mixture was incubated in the dark at about 37°C for 3 hours. A negative control (deionized water) was tested concurrently.
- Number of tissue replicates used per test chemical and controls: 2 replicates per test item/positive control/negative control. Freeze-killed control tissues were not tested as no direct reduction of MTT by the test substance occured.
- Wavelength used for quantifying MTT formazan: Measurement using a filter wavelength 570 nm without reference filter using a SunriseTM Absorbance Reader
- Description of the method used to quantify MTT formazan: The OD570 values determined for the various tissues are a measure of their viability. The ratio of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether a test material was an irritant.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: Please refer to table 1
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Please refer to table 2+3
- Reference to historical data of the RhCE tissue construct: Please refer to table 3
- Positive and negative control means and acceptance ranges based on historical data: Please refer to table 2+3
- Acceptable variability between tissue replicates for positive and negative controls: Please refer to table 2
- Acceptable variability between tissue replicates for the test chemical: Please refer to table 2

Results and discussion

In vitro

Results

Irritation parameter:

mean percent tissue viability 

Run / experiment:

Run 1 (mean of 2 replicate tissues)

Value:

2.5

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:

OTHER EFFECTS:
- Visible damage on test system: No

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: No

Any other information on results incl. tables

 

Table 4: Individual and mean OD570 values, individual and mean viability values and inter-tissue variability

Test substance identification		tissue 1	tissue 2	mean	Inter-tissue variability [%]
NC	mean OD570	2.300	2.383	2.342
	vability [% of NC]	98.2	101.8	100.0	3.5
Test item	mean OD570	0.071	0.045	0.058
	vability [% of NC]	3.0	1.9	2.5	1.1
PC	mean OD570	0.502	0.382	0.442
	vability [% of NC]	21.4	16.3	18.9	5.1

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Remarks:

The RhCE Test according to OECD guideline 492 showed an eye irritating potential of gamma-Valerolacton.