L-Glutamic acid, N-(15-carboxy-1-oxopentadecyl)-, 5-(2,5-dioxo-1-pyrrolidinyl) ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-07-28 to 2010-08-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study 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:

according to guideline

Guideline:

other: OECD 439 "In vitro skin irritation: Reconstituted Human Epidermis Model Test Method" (adopted 22 July 2010).

Deviations:

no

Principles of method if other than guideline:

NA

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

Species:

other: EPISKIN model

Strain:

other: EPISKIN model

Details on test animals or test system and environmental conditions:

NA

Test system

Type of coverage:

other: EPISKIN model

Preparation of test site:

other: EPISKIN model

Vehicle:

other: EPISKIN model

Controls:

other: EPISKIN model

Amount / concentration applied:

NA

Duration of treatment / exposure:

NA

Observation period:

NA

Number of animals:

NA

Details on study design:

The EPISKIN model is a three-dimensional reconstituted human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional corneum (SkinEthic Laboratories, Nice, France).

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. The concentration of the inflammatory mediator IL-1α in the culture medium was included as a complimentary end-point in the case of a borderline test result.

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, initially moistened with sterile water, were treated with 10 mg PC2414 for 15 minutes, followed by a post-exposure period of 42-hour. Triplicate tissues treated with a PBS solution served as negative control and triplicate tissues treated with 10 µl 5% (w/v) 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.

Results and discussion

In vivo

Irritant / corrosive response data:

Assessment of skin 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 negative control treated tissues appeared blue which was considered to be indicative of viable tissue. The positive control treated tissues appeared white which was considered to be indicative of dead tissue.

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

Any other information on results incl. tables

Table1. Mean OD540 values and percentage viabilities

Test substance	OD540	Mean OD540 ± SD	Relative viability (%)	Mean viability (%) ± SD
Negative Control	0.788	0.792 ± 0.023	99.5	100 ± 2.9
Negative Control	0.772		97.5
Negative Control	0.817		103.2
Positive Control	0.035	0.044 ± 2.3	4.4	5.6 ± 2.3
Positive Control	0.033		4.2
Positive Control	0.065		8.2
PC2414	0.717	0.826 ± 2.3	90.5	104.6 ± 12.0
PC2414	0.883		111.5
PC2414	0.878		110.9

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

quality controls for the EPISKIN 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.

Table 2. Qualitive evaluation of tissue viability (MTT uptake visual evaluation)

Material	Tissue 1	Tissue 2	Tissue 3
Negative Control Material	-	-	-
Positive Control Material	++	++	++
Test Material	-	-	-

MTT visual scoring scheme:

-     = blue tissue (viable)
+    = blue/white tissue (semi-viable)
++  = tissue is completely white (dead)

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

PC2414 was shown to be non-irritating to skin when tested in vitro in the EPISKIN model (SkinEthic Laboratories, Nice, France).

Executive summary:

The skin irritation potential of PC2424 was determined using the in vitro EPISKIN reconstituded human epidermis model (SkinEthic Laboratories, Nice, France) in accordance with OECD test guideline 439. Initially, a DEREK prediction of PC2414 did not indicate any toxicological effects including skin 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.