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Skin irritation / corrosion

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Endpoint:
skin irritation / corrosion
Remarks:
in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July 2015 to 18 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: pellets
Details on test material:
Identification: 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]
Appearance: White to off white pellet
Batch: T3J07001
Purity/Composition: 97.8%
Test substance storage: At room temperature
Stable under storage conditions until: 11 September 2017 (retest date)
Test substance handling: No specific handling conditions required
Chemical name (IUPAC): 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]
Trade name: NAUGARD® 412S
CAS Number: 29598-76-3
Molecular formula: C65H124O8S4
Molecular weight: 1161.94

In vitro test system

Test system:
human skin model
Source species:
other: human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Cell type:
non-transformed keratinocytes
Cell source:
other: cultured
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
Test system
EpiDerm Skin Model (EPI-200, Lot no.: 22299 kit O).
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 organized 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.

Rationale
Recommended test system in international guidelines (OECD and EC).

Source
MatTek Corporation, Ashland MA, U.S.A.

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 DMEM (Dulbecco’s Modified Eagle’s Medium)medium.

DMEM
Supplemented DMEM medium, serum-free supplied by MatTek Corporation.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) medium
MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation.

Environmental conditions
All incubations, with the exception of the test substance incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 66 - 85%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 36.7°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
30.5 to 35.1 mg of the solid test substance.
Duration of treatment / exposure:
3 minutes & 1 hour
Duration of post-treatment incubation (if applicable):
Not specified
Number of replicates:
2 tissues per test group (3 minutes exposure, 1 hour exposure, negative control, positive control).

Test system

Details on study design:
Test substance preparation
The correction factor is not applicable for the test, therefore no correction was made for the purity of the test item.
The solid test substance (30.5 to 35.1 mg) was crushed and ground in a mortar with pestle to improve the consistency and was applied directly on top of the skin tissue.

Reference substances
Negative control: Milli-Q water (Millipore Corp., Bedford, Mass., USA).
Positive control: Potassium hydroxide (KOH; Merck, Darmstadt, Germany), an 8.0 normal solution was prepared.

Study design
Test for the interference of the test substance with the MTT endpoint
A test substance may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test substance is present on the tissues when the MTT viability test is performed.

Test for colour interference by the test substance
The test substance was checked for possible colour interference before the study was started. Some non-coloured test substances may change into coloured substances in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of the test substance or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
In case the test substance induces colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test substance for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with DMEM medium.

Test for reduction of MTT by the test substance
2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] was checked for possible direct MTT reduction before the study was started. To assess the ability of the test substance to reduce MTT, approximately 25 mg of the test substance was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently. At the end of the exposure time it was checked if a blue / purple colour change was observed.
In case the test substance reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test substance and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT.

Application/Treatment of the test substance
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 approximately 1 hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM medium just before the test substance was applied. The test was performed on a total of 4 tissues per test substance together with a negative control and positive control. Two tissues were used for a 3-minute exposure to 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] and two for a 1-hour exposure. The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test substance to the tissue and 30.5 to 35.1 mg of the solid test substance was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test substance. 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 (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 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 substance was classified according to remaining cell viability following exposure of the test substance with either of the two exposure times.

Electronic data capture
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.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure
Value:
91
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 hour exposure
Value:
87
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

In vivo

Irritant / corrosive response data:
Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 91% and 87%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive.
Other effects:
The test substance was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because the solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test substance did not interfere with the MTT endpoint.

The absolute mean OD570 (optical density at 570 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 7%.

The maximum inter-tissue variability in viability between two tissues treated identically was less than 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%. It was therefore concluded that the test system functioned properly.

Any other information on results incl. tables

Mean absorption in the in vitro skin corrosion test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodeylthio)propionate]

 

3-minutes application

1-hour application

A (OD570)

B (OD570)

Mean (OD570)

 

SD

A (OD570)

B (OD570)

Mean (OD570)

 

SD

Negative control

2.041

1.991

2.016

±

0.035

2.208

2.026

2.117

±

0.128

Test substance

1.799

1.855

1.827

±

0.039

1.769

1.895

1.832

±

0.089

Positive control

0.152

0.132

0.142

±

0.014

0.197

0.237

0.217

±

0.028

SD = Standard deviation

Duplicate exposures are indicated by A and B.

In this table the values are corrected for background adsorption (0.0425). Isopropanol was used to measure the background adsorption.

 

Mean tissue viability in the in vitro skin corrosion test with 2,2-Bis[[3-(dodecylhio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate]

 

3-minute application viability (percentage of control)

1-hour application viability (percentage of control)

Negative control

100

100

Test substance

91

87

Positive control

7

10

 

INDVIDUAL OD MEASUREMENTS AT 570 NM

 

3-minute application (OD570)

1-hour application (OD570)

A

B

A

B

Negative control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

2.105

2.084

2.061

 

2.052

2.035

2.014

 

2.274

2.247

2.229

 

2.095

2.043

2.067

Test substance

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

1.872

1.822

1.830

 

1.900

1.903

1.889

 

1.834

1.810

1.790

 

1.954

1.939

1.918

Positive control

OD570measurement 1

OD570measurement 2

OD570measurement 3

 

0.193

0.195

0.197

 

0.175

0.176

0.174

 

0.238

0.244

0.237

 

0.282

0.277

0.281

OD = Optical density

Duplicate exposure are indicated by A and B.

 

HISTORICAL CONTROL DATA FOR IN VITRO SKIN CORROSION STUDIES

 

Negative control

Positive control

Positive control

3-minute treatment (OD570)

1-hour treatment (OD570)

3-minute treatment (OD570)

1-hour treatment (OD570)

3-minutes treatment (% viability)

1-hour treatment (% viability)

Range

1.076 – 2.167

1.361 – 2.203

0.017 – 0.29

0.063 – 0.226

6 – 16

4 – 12

Mean

1.78

1.78

0.15

0.12

10.6

7.0

SD

0.25

0.20

0.05

0.04

2.9

2.1

N

43

47

44

42

22

22

SD = Standard deviation

N = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period April 2012 to April 2015.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
2,2-Bis[[3-(dodecylthio)-1-xopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate] Naugard 412S is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

In vitro skin corrosion test with 2,2-Bis[[3-(dodecylthio)-1-oxopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], Naugard 412S, using a human skin model.

This report describes the ability of the test substance to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200). The possible corrosive potential of the test substance was tested through topical application for 3 minutes and 1 hour.

 

The study procedures described in this report were based on the most recent OECD and EC guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline no. 431: In Vitro Skin Corrosion: reconstructed human epidermis (RHE) test method (adopted 26 September 2014).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test". Official Journal of the European Union No. L142, 31 May 2008.

 

Batch T3J07001 of the test substance consisted of white to off white pellets with a purity of 97.8%. Skin tissue was moistened with 25 μl of Milli-Q water and approximately 25 mg of the test substance was applied directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 7% after 3 minutes exposure. The absolute mean OD570(optical density at 570 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 17% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 10%, indicating that the test system functioned properly.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance compared to the negative control tissues was 91% and 87%, respectively. Because the mean relative tissue viability for the test substance was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment the test substance is considered to be not corrosive.

 

Finally, it is concluded that this test is valid and that 2,2-Bis[[3-(dodecylthio)-1-xopropoxy]methyl]propane-1,3-diyl bis[3-(dodecylthio)propionate], Naugard 412S, is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.