Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 August 2011 to 31 October 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD Guideline No. 439
Deviations:
yes
Remarks:
see below
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 761/2009, B.46
Deviations:
yes
Remarks:
see below
Principles of method if other than guideline:
The study was performed in accordance with study plan No. 38014 TIC with the following deviations from the agreed study plan:
. during the treatment of the tissues with the positive control (SDS 5%), the tissue No. 3 was treated with a volume < 10 µL. As the acceptance criteria for the positive controls were fulfilled (viability of the tissue No. 3 is < 40% and the SD value between the tissue replicates viabilities are < 18%), this deviation was considered to not have compromised the validity or integrity of the study,
. during the test for direct MTT reduction, the test item was incubated with a freshly prepared MTT solution for 3 hours and 7 minutes instead of 3 hours ± 5 minutes. This deviation was considered to not have compromised the validity or integrity of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(piperidinothiocarbonyl) hexasulphide
EC Number:
213-537-2
EC Name:
Bis(piperidinothiocarbonyl) hexasulphide
Cas Number:
971-15-3
Molecular formula:
C12H20N2S8
IUPAC Name:
1,1'-(hexasulfane-1,6-diyldicarbonothioyl)dipiperidine
Constituent 2
Reference substance name:
Dipentamethylenethiuram hexasulfide
IUPAC Name:
Dipentamethylenethiuram hexasulfide
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstituted human epidermis
Cell source:
other: reconstituted human epidermis
Details on animal used as source of test system:
Episkin TM Model Kit (0.38 cm2 tissues) supplied by SkinEthic Laboratories, Lyon, France.
Medium and Incubation T°C: 37°C
Dates of experimental phase: from 30 August 2011 to 31 October 2011.
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
Amount/concentration applied:
As the test item was a solid, 5 µL of water for injection was first applied to each of the three tissues and then, 10 mg ± 2 mg of the test item were applied evenly to each tissue, taking care to spread it over the whole tissue surface without damaging the tissue sample.
Duration of treatment / exposure:
The test item, the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature during 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at 37°C, 5% CO2 in a humidified incubator.
Duration of post-treatment incubation (if applicable):
At the end of the 42-hour incubation period, the cell viability was then assessed by means of the colorimetric MTT reduction assay.
Number of replicates:
Tripicate tissues for each tested substance (test item, negative control, positive control)

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
treated
Value:
100.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Preliminary test
In the preliminary test, thetest item was found to not have direct MTT reducing properties since the MTTsolution containing the test item did notturn blue/purple when compared to the negative control.As a result, no additional controls were performed on water-killed tissues in parallel to the main test.
The test item was found to not have a coloring potential in the preliminary test since the water solution containing the test item did not change color. As a result, no additional controls were used in parallel to the main test.

Main test
All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered as valid.
Following a 15-minute exposure and a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 100.5%.

Any other information on results incl. tables

 Group Tissue No.  OD measurements (first) OD measurements (second) Mean OD blank  cOD first   cOD second Mean cOD   Viability (%)
 Negative control  1  1.033 1.004   0.996  0.967  0.982  96.0 
   2  1.089 1.054   0.037 1.052  1.017  1.035  101.2 
   3  1.108 1.108    1.071  1.032  1.052  102.8 
 Positive control  1  0.131 0.129    0.094  0.092  0.093  9.1 
   2  0.095 0.089  0.037  0.058 0.052  0.055  5.4 
   3  0.294 0.300    0.257  0.263  0.260  25.4 
 Test item  1  1.093 1.080    1.056  1.043 1.050  102.6 
   2  1.042 1.026  0.037  1.005  0.989 0.997  97.5 
   3  1.087  1.058   1.050  1.021  1.036  101.3 

OD = optical density

cOD = blanck corrected optical density

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
not skin irritating
Conclusions:
The test item is considered as non-irritant to skin in this in vitro test.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of the test item,Dipentamethylenethiuram hexasulfide,using the EpiskinTM reconstituted human epidermis model.

The study design is based on international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46) and thestudy was conducted in compliance with CIT’s standard operating procedures and the principles of Good Laboratory Practice.

Methods

Preliminary tests were performed to detect the ability of the test item to directly reduce MMT as well as its coloring potential.

Following the preliminary tests, the skin irritation potential of the test item was tested in one main test. The test item, the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature during 15 (± 1) minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 (± 1) hours at 5% CO2 in a humidified incubator. The cell viability was then assessed by means of the colorimetric MTT reduction assay.

Relative viability values were calculated for each tissue and expressed as percentages of the negative control tissues viability which was set at 100% (reference viability).

 

Results

Preliminary test

In the preliminary test, thetest item was found to not have direct MTT reducing properties since the MTTsolution containing the test item did notturn blue/purple when compared to the negative control.As a result, no additional controls were performed on water-killed tissues in parallel to the main test.

The test item was found to not have a coloring potential in the preliminary test since the water solution containing the test item did not change color. As a result, no additional controls were used in parallel to the main test.

Main test

All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered as valid.

Following a 15-minute exposure and a 42-hour recovery period, the relative mean viability of the tissues treated with the test item was 100.5%.

 

Conclusion

The test item is considered as non-irritant to skin.