Disodium 4,4'-methylenebis[3-hydroxy-2-naphthoate]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 MAY 2013 - 12 SEP 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Str. 80, 65189 Wiesbaden (1 Feb 2013)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 4,4'-methylenebis[3-hydroxy-2-naphthoate]

EC Number:

229-653-1

EC Name:

Disodium 4,4'-methylenebis[3-hydroxy-2-naphthoate]

Cas Number:

6640-22-8

Molecular formula:

C23H16O6.2Na

IUPAC Name:

disodium 4,4'-methylenebis(3-hydroxy-2-naphthoate)

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The RHE™-model was obtained by culturing adult human keratinocytes on a polycarbonate filter under conditions which permit their terminal differentiation and the reconstruction of an epidermis with a functional horny layer.

Justification for test system used:

To reduce animal testing, this alternative in vitro method was used. The human skin RHE™ model has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The applied human in vitro skin model RHE™ was produced by SkinEthic Laboratories (Lyon, France)
- Tissue batch number(s):

first run:
Batch No: 13 01U-M021
second run:
Batch No: 13 022A 0611

- Date of initiation of testing: 21 May 2013

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Using a multi pipette the tissues were gently rinsed with a minimum volume of 25 mL PBS to remove any residual test item. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h (± 5 min)
- Spectrophotometer: ELx800 Absorbance Microplate Reader, BioTek Instruments GmbH, Bad Friedrichshall, Germany
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
first run:
- Viability: OD = 1.463 (± 0.101) (Acceptance criterion: OD > 0.7)
- Barrier function: 4.7 h (Acceptance criterion: 4.0 h ≤ ET50 ≤ 9 h)
- Morphology: Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum. 5/6 viable cell layers present. Absence of significant histological abnormalities.

second run:
- Viability: OD=1.406 (±0.032) (Acceptance criterion: OD > 0.7)
- Barrier function: 6 h (Acceptance criterion: 4.0 h ≤ ET50 ≤ 9 h)
- Morphology: Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum. 5 viable cell layers present. Absence of significant histological abnormalities.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5 %

Duration of treatment / exposure:

42 min (± 1 min)

Duration of post-treatment incubation (if applicable):

42 h (± 1 h)

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

1

Value:

54.73

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

mean

Run / experiment:

2

Value:

77.75

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The visual evaluation in the pretest for MTT-reducing capacity of the test item after 3 hours incubation with MTT-reagent did not show blue color, i.e. MTT was not reduced by the test item.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control the absorbance values in both runs reached a mean OD of 1.695 (SD 2.07%) and 2.367 (SD 3.11%), respectively. Therefore, the negative control fulfilled the validity criteria.
- Acceptance criteria met for positive control: Treatment with the positive control in both runs revealed a mean relative viability of 1.59% (SD 5.01%) and 1.05% (SD 5.41%), respectively, thus the positive control reached the validity criteria.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of the negative control, the positive control and of the three tissues treated with the test item was ≤18%, respectively.

Any other information on results incl. tables

A table of the results is attached under 'Attached background material'.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The mean tissue viability after treatment with the test item was higher than 50% in both runs (first run: 54.73%, second run: 77.75%). Therefore, the test item is not considered to possess an irritant potential.

Executive summary:

Study Design

This GLP-compliant in vitro skin irritation study was performed according to OECD Guideline 439 (Reconstructed Human Epidermis Test). The test consisted of a topical exposure of the test item to a human reconstructed model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict skin irritation potential.

Triplicates of the human skin model RHE™ (Reconstructed Human Epidermis, SkinEthic Laboratories) were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (PBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before adding the solid test item, 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to the tissues. Due to the fact, that the first run with the test item revealed a borderline result (mean tissue viability: 54.73%) a second run was performed.

Results

Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) induced a sufficient decrease in the relative absorbance as compared to the negative control (PBS-buffer) for the treatment interval in both runs thus ensuring the validity of the test system. After treatment with the negative control (PBS-buffer) the absorbance values reached the required acceptability criterion of a mean optical density (OD) > 1.2 and < 2.5 for the treatment interval in both runs thus showing the quality of the tissues. Therefore, the study fulfilled the validity criteria. The mean tissue viability after treatment with the test item in the first run was 54.73%. Due to this borderline result of a mean percent viability equal to 50 ± 5%, a second run was performed according to OECD Guideline 439. The tissue viability after treatment with the test item in the second run was also higher than 50% (mean viability: 77.75%). Therefore, the test item is not considered to possess an irritant potential to the skin.

Conclusions

Under the conditions of the present study, the test item is not irritating to the skin.