4,4'-(2-ethylhexane-1,1-diyl)diphenol

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Valid GLP guideline study without deviations

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-(2-ethylhexane-1,1-diyl)diphenol

EC Number:

680-046-1

Cas Number:

74462-02-5

Molecular formula:

C20H26O2

IUPAC Name:

4,4'-(2-ethylhexane-1,1-diyl)diphenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: BisP-IOTD
Molecular formula: C20H26O2
Molecular weight: 298.43
CAS Number: 74462-02-5
Description: White powder
Batch: 120601
Purity/Composition: 99.6%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 12 March 2014 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)

Test animals

Species:

other: EPISKIN Small Model (EPISKIN-SM, 0.38 cm2, Batch no.1 13-EKIN-011)

Strain:

other: adult human-derived epidermal keratinocytes

Details on test animals or test system and environmental conditions:

EPISKIN Small Model ™ (EPISKIN-SM™, 0.38 cm2, Batch no.1 13-EKIN-011, received from SkinEthic Laboratories, Lyon, France). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
All incubations, with the exception of the test substance incubation of 15 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 63 - 98%), containing 5.0 ±0.5% CO2 in air in the dark at 37.0 ±1.0 °C (actual range 36.7 - 37.5 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the humidity (with a maximum of 17%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test system

Type of coverage:

other: not applicable

Preparation of test site:

other: not applicable

Vehicle:

water

Controls:

other: negative and positive controls were applied

Amount / concentration applied:

The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 µl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and the solid test substance (10.9 to 11.5 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 µl PBS (negative control) and 3 tissues with 25 µl 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 ml pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.

Duration of treatment / exposure:

After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 ml MTT-medium (0.3 mg/ml). The tissues were incubated for 3 h at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 µl isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN lnfinite® M200 Pro Plate Reader.

Observation period:

Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.
Observations/measurements in the study were recorded electronically using the following programme(s): REES Centron Environmental Monitoring system version SQL 2.0 (REES Scientific, Trenton, NJ, USA): Temperature and humidity. Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Number of animals:

All measurements (negative control, positive control and test substance) were performed in triplicates and results reported as mean

Details on study design:

Negative control: sterile Milli-Q water
Positive control: 5% aqueous sodium dodecyl sulphate solution (Sigma Aldrich) in phosphate buffered saline (PBS, Invitrogen Corp.)

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: viability

Value:

40

Remarks on result:

other:

Remarks:

Basis: mean of triplicates. Time point: following 15 minutes exposure. Max. score: 16.0. Reversibility: other: not applicable. Remarks: The max-score 16% is the viability observed with positive control. (migrated information)

In vivo

Irritant / corrosive response data:

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with BisP-IOTD compared to the negative control tissues was 40%. Since the mean relative tissue viability for BisP-IOTD was below 50% after 15 minutes treatment it is considered to be irritant. The positive control scored 16% in this test.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In this Reconstructed Human Epidermis Test Method assay the test substance BisP-IOTD was found being irritant to skin.

Executive summary:

BisP-IOTD was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that BisP-IOTD did not interact with MTT.

The mean absorption at 570 nm measured after treatment with BisP-IOTD and controls are presented as follows (mean value of double measurements of triplicate test sample):

Negative control: OD570 = 1.074 ±0.064 (set to 100% viability)

BisP-IOTD: OD570 = 0.432 ±0.087 (i.e. 40% of negative control)

Positive control: OD570 = 0.177 ±0.069 (16% of negative control)

Mean tissue viability obtained after 15 minutes treatment with BisP-IOTD compared to the negative control tissues is provided above. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with BisP-IOTD compared to the negative control tissues was 40%. Since the mean relative tissue viability for BisP-IOTD was below 50% it is considered to be irritant.

The positive control had a mean cell viability after 15 minutes exposure of 16%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 9%, indicating that the test system functioned properly.