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

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 August 2013 - 15 October 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

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
- Source and lot/batch No.of test material: E00031-633

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the dark at ambient temperature

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Justification for test system used:

The EpiSkin RhE model has been accepted as a valid model for the assessment of skin irritation by European Centre for the Validation of Alternative Methods (ECVAM) and the Organisation for Economic Co-operation and Development (OECD).

Vehicle:

unchanged (no vehicle)

Details on test system:

The EpiSkin RhE tissues were produced by culturing adult human keratinocytes on a collagen base in conditions which permitted their terminal differentiation and the reconstruction of an epidermis with a functional horny layer (stratum corneum). The human keratinocytes came from mammary samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2, HEP B and HEP C tests were carried out on the donors as well as verification of the bacteriological and fungal sterility of the cells and absence of mycoplasma. This model is used to assess the irritation potential of a test item by examining the initiating events in the cascade, for example: the cytotoxicty. The test system measures the reduction of MTT to formazan metabolite by mitochondrial reductase and, therefore, irritant materials are identified by their ability to reduce cell viability below a threshold of 50% below the negative control value.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL (Dulbecco's PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL (aqueous SDS solution (5%, w/v))

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42.5 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 99.9

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:

- OTHER EFFECTS:
- Visible damage on test system: No visible damage
- Direct-MTT reduction: yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
Acceptance criteria: OD: >/=0.6 and - Acceptance criteria met for positive control: yes
Acceptance criteria: mean % viability: - Acceptance criteria met for variability between replicate measurements: yes

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

EXP0700332 is non-irritant when tested in the EpiSkin in vitro irritiation assay

Executive summary:

The dermal irritation potential was assessed by applying EXP0700332 (10 µL) to the exposed surface of three EpiSkin reconstructed human epidermis (RhE) units for 15 min. The surface area of the EpiSkin was 0.38 cm^2, therefore the application rate was 26.3 µL/cm^2. After the 15 min exposure period, the surface of the EpiSkin was rinsed with Dulbecco’s phosphate-buffered saline (PBS; 25 mL). Initial rinsing with PBS did not remove all of the applied dose from the EpiSkin surface. Therefore, cotton swabs soaked in PBS were used to gently remove any remaining test item from the surface of the EpiSkin units. The EpiSkin was then incubated in maintenance media (2 mL) for a recovery period of 42.5 hours in a humidified incubator set to maintain temperature and CO2 levels of 37 degrees Celsius and 5%, respectively. Following incubation, the EpiSkin units were transferred to assay medium (2 mL) containing MTT (0.3 mg/mL) and returned to the incubator (set to maintain temperature and CO2 levels of 37 degrees Celsius and 5%, respectively) for 3 hours. Biopsies of the EpiSkin membranes were removed, added to acidified isopropanol and refrigerated for ca 68 h in order to extract the formazan. 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, aqueous sodium dodecyl sulphate (SDS) solution (5%, w/v), and the negative control, PBS, were tested in parallel to demonstrate the efficacy of the assay. The viability of each individual EpiSkin tissue was calculated as a percentage of the mean negative control viability (defined as 100%). Exposure to EXP0700332 resulted in a mean EpiSkin viability of 99.90% +/- 9.42% of the negative control value. Exposure to the positive control, aqueous SDS solution (5%, w/v), resulted in a mean EpiSkin viability of 10.62% +/- 3.54% of the negative control value.