4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 July 2012 to 27 August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidance in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

EC Number:

217-420-7

EC Name:

4,4',4''-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

Cas Number:

1843-03-4

Molecular formula:

C37H52O3

IUPAC Name:

4-[4,4-bis(5-tert-butyl-4-hydroxy-2-methylphenyl)butan-2-yl]-2-tert-butyl-5-methylphenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]
Molecular formula: C37H52O3
Molecular weight: 544.81
CAS Number: 1843-03-4
Description: White powder (determined at NOTOX)
Batch: WCA1A0001
Purity: 97.6 Area%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 09 October 2012 (Retest date)

Test animals

Species:

other: EPISKIN Small Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Test system: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-30).
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.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Source: SkinEthic Laboratories, Lyon, France.

Test system

Type of coverage:

other: Detailed under study design

Preparation of test site:

other: Detailed under study design

Vehicle:

unchanged (no vehicle)

Controls:

other: negative and positive control performed

Amount / concentration applied:

11.0 to 14.3 mg

Duration of treatment / exposure:

15 minutes

Observation period:

42-hour post incubation period.

Number of animals:

Not applicable. Triplicate exposure were performed.

Details on study design:

Test substance preparation: No correction was made for the purity/composition of the test compound.
The solid test substance (11.0 to 14.3 mg) was applied directly on top of the skin tissue. 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was spread to match the size of the tissue.

Reference substances
Negative control:Phosphate buffered saline (PBS, Invitrogen Corporation, Breda, The Netherlands).
Positive control:5% (aq) Sodium dodecyl sulphate (SDS, Sigma Aldrich, Zwijndrecht, The Netherlands) [CAS Number 151-21-3] in PBS.

Preparation and preincubation
Tissues: On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 24 hours at 37°C. Maintenance medium and Assay medium were supplied by Skinethic Laboratories, Lyon, France.

MTT medium: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).

Environmental conditions: 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 83 - 96%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 37.0 - 37.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Study design
Test for reduction of MTT by the test substance: 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, 12.6 mg of the test substance was added to 2 ml MTT solution (0.3 mg/ml in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently.

Application/Treatment of the test substance: 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 (11.0 to 14.3 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.

Cell viability measurement: 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 approximately 70 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.

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.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Mean adsorption

Value:

0.886

Remarks on result:

other:

Remarks:

Basis: mean. Time point: overall. Max. score: 0.893. Remarks: SD +/- 0.042. (migrated information)

In vivo

Irritant / corrosive response data:

4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] 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 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] did not interact with MTT.

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 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] compared to the negative control tissues was 119%. Since the mean relative tissue viability for 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was above 50% 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is considered to be non-irritant.

The positive control had a mean cell viability after 15 minutes exposure of 6%. The standard deviation value of the percentage viability of three tissues treated identically with the positive control and test substance was less than 5%. Although the SD of the percentage viability of three tissues treated identically with the negative control was 19% , the absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Mean absorption in the in vitro skin irritation test with 4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.750	0.853	0.576	0.727	±	0.140
Test substance	0.893	0.817	0.888	0.866	±	0.042
Positive control	0.045	0.057	0.024	0.042	±	0.016

Test substance = 4,4’,4’’-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol]

OD = optical density

SD = standard deviation

Triplicate exposure are indicated by A, B and C.

In this table the values are corrected for background absorption (0.042). Isopropanol was used to measure the background absorption.

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

In vitro skin irritation test with 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] using a human skin model.

 

This report describes the ability of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small Model (EPISKIN-SMTM)). The possible skin irritation potential of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was tested through topical application for 15 minutes.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines and performed in compliance with the Principles of Good Laboratory Practice.

 

Batch WCA1A0001 of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was a white powder with a purity of 97.6 Area%. Skin tissue was moistened with 5 µl of Milli-Q water and 11.0 to 14.3 mg of 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was applied directly on top of the skin tissue for 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

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 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] compared to the negative control tissues was 119%. Since the mean relative tissue viability for 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] was above 50% after 15 minutes treatment 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is considered to be non-irritant.

 

The positive control had a mean cell viability of 6% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically with the positive control and test substance was less than 6%. Although the SD of the percentage viability of three tissues treated identically with the negative control was 19%, the absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

Finally, it is concluded that this test is valid and that 4,4’,4”-(1-methylpropanyl-3-ylidene)tris[6-tert-butyl-m-cresol] is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.