6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 April 2017 to 13 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for the Testing of Chemicals, No. 431, (29 July 2016) “In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method”

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test

Version / remarks:

Commission Regulation (EC) No 440/2008, Annex Part B, B.40.Bis: “In Vitro Skin Corrosion: Human Skin Model Test”, Official Journal of the European Union No. L142 (31 May 2008)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

EC Number:

202-009-7

EC Name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Cas Number:

90-66-4

Molecular formula:

C22H30O2S

IUPAC Name:

6,6'-di-tert-butyl-2,2'-thiodi-p-cresol

Test material form:

solid: particulate/powder

Details on test material:

Name: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol
Other name: LOWINOX® TBP-6
CAS number: 90-66-4
Batch/Lot Number: C034J0059 / C036K0111
Description: White powder
Purity*: 99.8%
Expiry date: 12 May 2017
Storage condition: Controlled room temperature (15-25 ºC, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
* No correction for purity of the test item was applied.

Specific details on test material used for the study:

No further details specified in the study report.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

EPISKIN TM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-015, Expiry Date: 17 April 2017) is a three-dimensional human epidermis model. Adult human derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKIN TM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

INDICATOR FOR POTENTIAL FALSE VIABILITY
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis.
If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.

Check-method for possible direct MTT reduction with test item
20 mg of test item was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours and then any colour change was observed:
-Test items which do not react with MTT: yellow
-Test items reacting with MTT: blue or purple
After three hours of incubation, yellow colour of the mixture was detected; therefore additional controls were not used in the experiment.

Check-method to detect the colouring potential of test item
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSCliving %) was determined in order to evaluate the ability of test item to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test item-treated living tissues were used for the non specific OD evaluation. These tissues followed the same test item application and all steps as for the other tissues, except for the MTT step:
MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD readings were conducted following the same conditions as for the other tissues.

PERFORMANCE OF THE STUDY
Pre-incubation (Day [-1])
The Maintenance Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37°C in an incubator with 5% CO2 in a >95% humidified atmosphere.

Application (Day 0)
The Assay Medium was pre-warmed to 37°C. The appropriate number of wells in an assay plate was filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, whereby each epidermis was in contact with the medium in the corresponding well underneath. Two epidermis units were used for each test or control materials.
- 20 mg of test item was applied evenly to the epidermal surface of each of two test units and each additional control skin units and then 100 μL physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.9-24.6°C) covered with the plate lids.

Rinsing (Day 0)
After the incubation time (4 hours), all test item treated tissues or also the positive control tissues were removed and rinsed thoroughly with PBS solution to remove all the remaining test or positive control material from the epidermal surface. Likewise, negative control tissues were processed accordingly.
The rest of the PBS was removed from the epidermal surface using a pipette (without touching the epidermis).

MTT test (Day 0)
MTT solution (2 mL of 0.3 mg/mL MTT working solution) was added to each well below the skin units (except of the two living colour control units). The lid was replaced and the plate incubated at 37°C in an incubator with 5% CO2 for 3 hours (±15 minutes), protected from light.

Formazan extraction (Day 0)
At the end of incubation with MTT a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this procedure involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated overnight at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
A blank sample containing 2 mL of acidified isopropanol was processed in parallel.

Cell viability measurements (Day 1)
Following the formazan extraction, 2×200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 22 August 2016, calibration is valid until August 2018) at the required wavelength on each day before use.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 mg of test item was applied evenly to the epidermal surface of each of two test units and each additional control skin units and then 100 μL physiological saline was
added to the test item to ensure good contact with the epidermis.

Duration of treatment / exposure:

Single exposure

Duration of post-treatment incubation (if applicable):

The plates with the treated epidermis units were incubated for 4 hours (±10 min) at room temperature (23.9-24.6°C) covered with the plate lids.

Number of replicates:

Two epidermis units were used for each test or control materials.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

87.4

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:

ADDITIONAL CONTROLS
As the test item was coloured, two additional test item-treated tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.003, Non Specific Colour% (NSCliving%) was calculated as 0.3% (see Table 1). This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.
As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary to exclude the false estimation of viability.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 2. The mean OD value for the test item treated skin samples showed a 87.4% relative viability.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (1.070).
The two positive control treated tissues showed 0.5% viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 20.2%.
The difference of viability between the two negative control tissue samples in the MTT assay was 1.3%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

Additional control	Optical Density (OD)			NSC% (living)
		Measured	Blank corrected
Treated with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol	1	0.049	0.003	0.3
	2	0.049	0.003
	Mean	--	0.003

Notes:

1. Mean blank value was 0.046

2. Optical density means the mean value of the supplicate wells for each sample (rounded to three decimal places).

 

Optical Density (OD) and the calculated relative viability % of the samples

Substance	Optical Density (OD)			Viability (% RV)
		Measured	Blank corrected
Negative Control: Physiological saline (0.9% (w/v) NaCl)	1	1.123	1.077	100.7
	2	1.109	1.063	99.3
	Mean	--	1.070	100.0
Positive Control: Glacial acetic acid	1	0.053	0.007	0.6
	2	0.050	0.004	0.3
	Mean	--	0.005	0.5
Test Item: 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol	1	1.076	1.030	96.3
	2	0.887	0.841	78.6
	Mean	--	0.935	87.4

Notes:

1. Mean blank value was 0.046

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places)

HISTORICAL CONTROL DATA

	Negative control (Physiological saline)	Positive control (Glacial acetic acid)
Minimum optical density (OD)	0.611	0.005
Maximum optical density (OD)	1.516	0.051
Mean optical density (OD)	0.871	0.017
Standard Deviation (SD)	0.164	0.010
Number of cases	81	81

Note: All optical density (OD) values measured are background corrected values (measured at 570 ± 30 nm)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in this in vitro EPISKIN™(SM) model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non corrosive to the skin, UN GHS Classification: No Category.

Executive summary:

An in vitro skin corrosivity test of 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

 

Disks of EPISKINTM(SM) (two units) were treated with 6,6’-di-tert-butyl-2,2’-thiodip-cresol test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

 

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

 

Following exposure with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol, the mean cell viability was 87.4% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

 

In conclusion, in this in vitro EPISKIN™(SM) model test with 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol (Batch number: C034J0059), the results indicate that the test item is non corrosive to the skin, UN GHS Classification: No Category.