Condensation products of Dihydrofuran-2,5-dione, mono-alkenyl derivs. with polyethylene amine and acetic anhydride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2013 to 07 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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)

Deviations:

yes

Remarks:

Study performed before the guideline came into force.

Principles of method if other than guideline:

Evaluation of ocular irritation is part of the Human Health Hazard Assessment required for registration of a chemical. In this study, the irritation potential of EXP0700332 was assessed using the HCE® in vitro ocular irritation test.

The SkinEthic HCE® model assesses the irritation potential of a test item by examining the cytotoxic effects of the test item after a defined exposure period and recovery time. The endpoint of the HCE® assay is the estimation of cell viability by assaying the reduction of MTT by mitochondrial enzymes. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.

30 µL of EXP0700332, the postitive control and the negative control were applied directly onto the surface of HCE tissue using a positive displacement pipette.The tissue was exposed for 60 minutes before rinsing with PBS (25 mL). After washing, all HSE tissues were transferred to fresh maintenance medium and incubated for 18 hours at 5% CO2 and 37 degrees Celsius. The tissues were then transferred to a new 24 well plate containing MTT solution in SkinEthic maintenance medium (0.5 mg/mL, 300 µL per well). The HCE® tissues were then incubated for 3 hours in a humidified incubator set to maintain temperature and CO2 levels of 37 degrees Celsius and 5%, respectively. The cells were then removed from the MTT solution and gently dried before being transferred to wells containing 750 µL of isopropanol to extract the formazan for 90 minutes. The absorbance was then measured to determine the cell viability.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: extremely viscous amber liquid
- Analytical purity:
- Lot/batch No.: E00031-68-1
- Expiration date of the lot/batch: 16 October 2010
- Storage condition of test material:room temperature in the dark, under nitrogen

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: E00031-633
- Retest date of the lot/batch: 30 January 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark

Test animals / tissue source

Species:

human

Strain:

other: Human Corneal Epithelium

Details on test animals or tissues and environmental conditions:

Evaluation of ocular irritation is part of the Human Health Hazard Assessment required for registration of a chemical. In this study, the irritation potential of EXP0700332 was assessed using the HCE® in vitro ocular irritation test.
The SkinEthic reconstructed Human Corneal Epithelium (HCE) model has undergone ECVAM prevalidation testing and has been shown to have a high correlation with existing in vivo and in vitro data for known eye irritants and non-irritant compounds.

The SkinEthic HCE® model assesses the irritation potential of a test item by examining the cytotoxic effects of the test item after a defined exposure period and recovery time. The endpoint of the HCE® assay is the estimation of cell viability by assaying the reduction of MTT by mitochondrial enzymes. Irritant materials are identified by their ability to reduce cell viability below a threshold of 50% of the negative control value.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 µL (application rate of 60 µL/cm2)

Duration of treatment / exposure:

60 minutes

Observation period (in vivo):

Not Applicable

Duration of post- treatment incubation (in vitro):

After exposure of the test substance, the HCE was washed with 25 mL PBS and incubated for 18 hours at 37 degrees celsius at 5% CO2.

Number of animals or in vitro replicates:

3 replicates per treatment

Details on study design:

EXP0700332, the positive control (Triton X-100 solution; 20%, w/v) and negative control (PBS), were each applied directly (30 µL) onto the surface of four HCE tissues using a positive displacement pipette. The pipette tip was used to gently spread the applied doses over the entire exposed area of the HCE tissues. The surface area of the EpiSkin was 0.5 cm2. Therefore, the mean application rate was 60 µL/cm2 for all treatments.

After exposure to the test item and controls for 60 minutes, the exposure period was terminated by rinsing the HCE® tissues with PBS (25 mL). After washing, all HCE® tissues were transferred to fresh maintenance medium and incubated for 18 hours in a humidified incubator set to maintain temperature and CO2 levels of 37 degrees Celsius and 5%, respectively.

After the recovery period, the HCE® tissues were transferred to a new 24 well plate containing MTT solution in SkinEthic maintenance medium (0.5 mg/mL, 300 µL per well). The HCE® tissues were then incubated for 3 hours in a humidified incubator set to maintain temperature and CO2 levels of 37 degrees Celsius and 5%, respectively. After the incubation, each HCE® tissue was removed from the MTT solution and gently tapped dry to remove any excess moisture. The HCE® tissues were then transferred to wells containing 750 µL isopropanol to extract the formazan. A further 750 µL of isopropanol was also added to the upper surface of each HCE® tissue. The wells were then sealed and left for 90 minutes at room temperature, protected from light, with gentle shaking. Following the solvent extraction two aliquots were added to a 96 well plate for each sample. Plates were analysed using an MRX plate reader using wavelength measurement at 550 nm. Absorbance values were calculated against the background isopropanol sample contained on the plate.

Results and discussion

In vitro

Results

Irritation parameter:

other: Cell viability (%)

Value:

ca. 100.81

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

EXP0700332 was demonstrated to be non-irritant to eyes when tested in vitro using the HCE® reconstructed human corneal model.

Executive summary:

Evaluation of ocular irritation is part of the Human Health Hazard Assessment required for registration of a chemical. In this study, the irritation potential of EXP0700332 was assessed using the HCE® in vitro ocular irritation test.

Prior to the conduct of the irritation assay, a preliminary test was conducted to assess the intrinsic ability of the test item to reduce methylthiazoldiphenyl-tetrazolium bromide (MTT) to formazan, which is a measure of cell viability in the assay. The results of the MTT direct reduction test showed that EXP0700332 did not reduce MTT to formazan.

Eye irritation potential was assessed by applying EXP0700332 (30 µL) to the exposed surface of three HCE tissues for 60 minutes. The surface area of the HCE was 0.5 cm2, therefore the application rate was 60 µL/cm^2. After the 60 minutes exposure period, the test item was washed from the surface of the HCE using Dulbecco’s phosphate-buffered saline (PBS; 25 mL) and cotton swabs. The HCE tissues were then incubated for a recovery period of 16 hours in a humidified incubator set to maintain temperature and CO2 levels of 37 degrees Celsius and 5%, respectively. Following incubation, the HCE tissues were transferred to maintenance medium containing MTT (0.5 mg/mL) and returned to the incubator for 3 hours. The HCE tissues were then transferred to isopropanol in order to extract the formazan. After extraction (90 minutes), the formazan production (cell viability) was assessed by measuring the optical density of the extracts at a wavelength of 550 nm. Three replicates of the positive control,

Triton X-100 solution (20%, w/v), and the negative control, PBS, were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual HCE tissue was calculated as a percentage of the mean negative control viability (defined as 100%).

Exposure to EXP0700332 resulted in a mean HCE® viability of 100.81 +/- 7.23% of the negative control value. Exposure to the positive control, Triton X-100 (20%, w/v), resulted in a mean HCE® viability of 0.23 +/- 0.11% of the negative control value. Cell viability values below a threshold of 50% of the negative control viability indicate that the test item is irritant.

In conclusion, EXP0700332 was demonstrated to be non-irritant to eyes when tested in vitro using the HCE® reconstructed human corneal model.