16-[[(1S)-1-carboxy-4-(2,5-dioxopyrrolidin-1-yl)oxy-4-oxo-butyl]amino]-16-oxo-hexadecanoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-09-08 to 2010-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed as an in vitro study using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France). This type of study, although not yet formally validated or adopted by regulatory authorities usually is a stand-alone test or as part of a tiered testing strategy for determining eye irritation at the lower end of irritancy. Initially, a DEREK prediction of PC2414 did not indicate any toxicological effects including eye irritation, indicating the need for a tiered testing strategy. The study protocol used has undergone successful pre-validation at Harlan Laboratories Ltd. and will form part of an international validation study during 2010 designed to offer a replacement to the in vivo rabbit eye test. The study was performed in accordance with GLP regulation and with no deviation to study plan.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2011

Materials and methods

Test guideline

Qualifier:

no guideline followed

Deviations:

not applicable

Principles of method if other than guideline:

The purpose of the study was to determine the eye irritation potential of PC2424 using the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France). The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the PC2414 treated tissues (quantitative measurement of tissue viability) relative to the negative control. Based on the relative mean tissue viability, a chemical is considered irritant (I) if the tissue viability is less than 60% and non-irritant (NI) if the tissue viability is equal or larger than 60%.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

PC2414

IUPAC Name:

PC2414

Details on test material:

- Name of test material (as cited in study report): PC2414
- Lot/batch No.: Y5UB376
- Expiration date of the lot/batch: 2013-03-01

Test animals / tissue source

Species:

other: SkinEthic Reconstituted Human Corneal mode

Strain:

other: SkinEthic Reconstituted Human Corneal mode

Details on test animals or tissues and environmental conditions:

NA

Test system

Vehicle:

other: SkinEthic Reconstituted Human Corneal mode

Controls:

other: SkinEthic Reconstituted Human Corneal mode

Amount / concentration applied:

NA

Duration of treatment / exposure:

NA

Observation period (in vivo):

NA

Number of animals or in vitro replicates:

NA

Details on study design:

The SkinEthic HCE model consists of transformed human keratinocytes of the cell line HCE (LSU EYE Center, New Orleans, USA) that form a corneal epithelial tissue (mucosa), devoid of stratum corneum, resembling, historically, the mucosa of the human eye. PC2414 was applied directly to the culture surface, at the air interface, so that undiluted and/or end use dilutions can be tested directly. The model consists of an airlifted, living, corneal tissue construct, produced in polycarbonate inserts in serum-free and chemically defined medium.

The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers.

Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the PC2414 treated tissues (quantitative measurement of tissue viability) relative to the negative control. PC2414 treated tissue was retained for further histopathological investigations if indicated in the cytoxicity determination.

The experimental design of the study consisted of a test for direct reduction of MTT by PC2414 followed by the main test. In the main test, triplicate tissues were treated with 30 mg PC2414 for 10 minutes. Triplicate tissues treated with a standeard solution A served as negative control and triplicate tissues treated with 30 ul 1% SDS served as positive control.

At the end of the exposure period each tissue was rinsed. The rinsed tissues (two per group) were taken for MTT loading. The remaining tissues were retained for possible histopathology.

Following MTT loading the reduced MTT was extracted from the tissues. After extraction the absorbency of triplicate aliquots of the extracted MTT solution for each tissue was measured. The optical density was measured at 540 nm (OD540). The percentage viability was calculated as MTT conversion relative to the negative controls.

The tissues retained for possible histopathology were carefully cut out of the polycarbonate inserts with a sharp scalpel. The tissues were carefully cut in half. Both halves were placed into a pre-labelled 1.5 ml Eppendorf tube containing 1 ml of 10% Formalin and stored at room temperature.

Results and discussion

In vivo

Irritant / corrosive response data:

Assessment of eye irritation potential (Table 1):
The relative mean viability of PC2414 after a 10 min exposure period was 71.1%. The relative mean viability of the positive control after a 10 min exposure period was 45.1%..

Other effects:

PC2414 and positive control treated tissues appeared blue/white which was considered to be indicative of semi-viable tissue. The negative control treated tussies appeared blue which was considered to be indicative of viable tissue. Visual assessment is a subjective observation incorporated for trhe purpose of identifying possible equivocal results. The results of the visual observation is approximate and has no impact on the classification of PC2414.

Based on the results it was considered unnecessary to proceed with tissue histopathalogy.

Any other information on results incl. tables

Table 1. Assessment of eye irritation of potential of PC2414 - viability of HCE tissues

Test substance	OD540	Mean OD540	Relative viability (%)
Negative Control	0.977	0.983	100
Negative Control	0.989
Positive Control	0.442	0.443	45.1
Positive Control	0.443
PC2414	0.643	0.699	71.1
PC2414	0.754

The relative viability of the positive and negative controls groups were appropriate and the assay acceptance criteria fulfilled. The

quality controls for the HCE model was acceptable according to the supplied technical data sheet and together with the fulfilled assay acceptance criteria the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

PC2414 was shown not to be non-irritating to eye when tested in vitro in the SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France).

Executive summary:

The eye irritation potential of PC2424 was determined using the in vitro SkinEthic Reconstituted Human Corneal model (HCE, SkinEthic Laboratories, Nice, France). This type of study, although not yet formally validated or adopted by regulatory authorities was part of a tiered testing strategy for determining eye irritation at the lower end of irritancy. Initially, a DEREK prediction of PC2414 did not indicate any toxicological effects including eye irritation, indicating the need for a tiered testing strategy.

The test is based on the hypothesis that irritant chemicals are able to penetrate the stratum corneum of the SkinEthic HCE model and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Cytotoxicity was determined by the reduction of MTT to formazan by viable cells in the PC2414 treated tissues (quantitative measurement of tissue viability) relative to the negative control. In this test, the quality criteria required for acceptance of results were satisfied and PC2414 was determined to be non-irritant.