Phenol, C14-18-alkyl derivs.

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August 2014 to 29 August 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test"

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

C14-16-18 Alkyl phenol

EC Number:

931-468-2

Cas Number:

1190625-94-5

Molecular formula:

For substance information - see Section 1.2

IUPAC Name:

C14-16-18 Alkyl phenol

Test material form:

other: slightly viscous liquid

Details on test material:

- Appearance: Clear amber liquid
- Storage conditions of test material: At room temperature in the dark

Test animals

Species:

other: EpiDerm Skin Model

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

TEST SYSTEM
EpiDerm Skin Model (EPI-200, Lot no.:20565 Lot R, MatTek Corporation, Ashland MA, USA).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Test system

Type of coverage:

open

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

other: tissues were exposed to both positive (8 N potassium hydroxide) and negative (Milli-Q water) concurrent controls

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL. The liquid test material was applied undiluted directly on top of the tissue.

Duration of treatment / exposure:

3 minutes and 1 hour

Observation period:

After exposure the skin tissue was thoroughly rinsed to remove the test material, followed by determination of the cytotoxic (corrosive) effect.

Number of animals:

Two tissue samples were exposed for 3 minutes and two were exposed for 1 hour.

Details on study design:

CELL CULTURE
- Tissues: On the day of receipt the tissues were kept on agarose and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 mL Dulbecco’s Modified Eagle’s Medium (supplemented DMEM medium, serum-free).
- MTT medium: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) concentrate (5 mg/mL) diluted (1:5) with MTT diluent (supplemented DMEM).
- Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 to 100 % (actual range 68 to 84 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.3 to 36.6 °C).

TEST FOR REDUCTION OF MTT BY THE TEST MATERIAL
The test material was checked for possible direct MTT reduction before the study was started. To assess the ability of the test material to reduce MTT, 100 µL of the test material was added to 1 mL MTT medium. The mixture was incubated for approximately 1 hour at room temperature in the dark. A negative control, sterile Milli-Q water was tested concurrently.

APPLICATION/TREATMENT OF THE TEST MATERIAL
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM medium per well. The level of the DMEM medium was just beneath the tissue. The plates were incubated for 3 hours at 37.0 ± 1.0 °C. The medium was replaced with fresh DMEM medium just before the test material was applied. The undiluted test material was added with a pipette.
After the exposure period, the tissues were washed with phosphate buffered saline (PBS) to remove residual test material. Rinsed tissues were kept in 24 well plates on 300 µL DMEM medium until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM medium was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37 °C in air containing 5 % CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol over night at room temperature.
The amount of extracted formazan was determined spectrophotometrically at 540 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues.
Skin corrosion potential of the test material was classified according to remaining cell viability following exposure of the test material with either of the two exposure times.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: Relative mean tissue viability

Value:

92

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minute exposure. Max. score: 100.0. Reversibility: no data. Remarks: The value was 92 %. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: Relative mean tissue viability

Value:

89

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 hour exposure. Max. score: 100.0. Reversibility: no data. Remarks: The value was 89 %. (migrated information)

In vivo

Irritant / corrosive response data:

The test material was checked for possible direct MTT reduction by adding it to MTT medium. As no colour change was observed, it was concluded that the test material did not interact with MTT.

The mean absorption at 540 nm measured after treatment with the test material and controls are presented in Table 1. Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues.
Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 92 % and 89 %, respectively. As the mean relative tissue viability was not below 50 % after 3 minutes of treatment and not below 15 % after 1 hour of treatment, the test material is considered to be not corrosive.
The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 8 %. The maximum inter-tissue variability in viability between two tissues treated identically was less than 4 % and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 2 %. It was therefore concluded that the test system functioned properly.

Any other information on results incl. tables

Table 1: Mean absorption in the in vitro skin corrosion test

	3- Minute Application			1-Hour Application
	A (OD540)	B (OD540)	Mean ± SD	A (OD540)	B (OD540)	Mean ± SD
Negative Control	1.823	1.768	1.796 ± 0.039	1.858	1.859	1.859 ± 0.001
Test Material	1.681	1.618	1.650 ± 0.044	1.656	1.665	1.660 ± 0.006
Positive Control	0.145	0.146	0.146 ± 0.001	0.130	0.127	0.128 ± 0.002

SD = Standard deviation

Duplicate exposures are represented by A and B

Values are corrected for background absorption (measured using isopropanol)

 

Table 2: Mean tissue viability in the in vitro skin corrosion test

	3- Minute Application Viability (% of control)	1-Hour Application Viability (% of control)
Negative Control	100	100
Test Material	92	89
Positive Control	8	7

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.

Executive summary:

The corrosion potential of the test material was investigated in an in vitro skin corrosion test using a human skin model in accordance with the standardised guidelines OECD 431 and EU Method B.40 BIS under GLP conditions.

The study determined the ability of the test material to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential was tested through topical application for 3 minutes and 1 hour. The test material was applied undiluted (50 µL) directly on top of the skin tissue. Tissues were also exposed to both positive (8 N potassium hydroxide) and negative (Milli-Q water) concurrent controls.

Skin corrosion is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test material compared to the negative control tissues was 92 % and 89 %, respectively. As the mean relative tissue viability for the test material was not below 50 % after the 3-minute treatment and not below 15 % after the 1-hour treatment, it is considered to be not corrosive.

The positive control had a mean relative tissue viability of 8 % after 3 minutes of exposure. The absolute mean OD540 (optical density at 540 nm) of the negative control tissues was within the laboratory historical control data range. The maximum inter-tissue variability in viability between two tissues treated identically was less than 4 % and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 2 %, indicating that the test system functioned properly. It was concluded that the test was valid.

Under the conditions of this study, the test material is not corrosive in the in vitro skin corrosion test.