Dipentylammonium dipentyldithiocarbamate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February 2018 to ****

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Carbamodithioic acid, dipentyl-, branched and linear, compds. with branched and linear N-pentyl-1-pentanamine

EC Number:

294-061-2

EC Name:

Carbamodithioic acid, dipentyl-, branched and linear, compds. with branched and linear N-pentyl-1-pentanamine

Cas Number:

91672-53-6

Molecular formula:

C21H46N2S2

IUPAC Name:

Branched and linear N-pentylpentan-1-aminium branched and linear dipentyldithiocarbamate

Specific details on test material used for the study:

Purity: 100%
Storage: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: reconstructed human epidermis tissues

Cell source:

other: No specified

Vehicle:

unchanged (no vehicle)

Details on test system:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT in the pre-test for direct MTT reduction and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues.

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre labeled 96 well plate. The optical density was measured at 570 nm (OD570).

Control samples:

other: Negative Control: Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ and Positive Control: Sodium Dodecyl Sulphate (SDS)

Amount/concentration applied:

10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface.

Duration of treatment / exposure:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period each tissue was rinsed before incubating for 42 hours.

Number of replicates:

Three

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

using the EPISKINTM reconstructed human epidermis model after a treatment period of 15 minutes followed by a post exposure incubation period of 42 hours

Value:

101.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Test for Direct MTT Reduction

The MTT solution containing the test item turned purple which indicated that the test item directly reduced MTT. Therefore, an additional procedure using water-killed tissues was performed. However, the results obtained showed that no effects on OD₅₇₀values (Appendix 2) that indicated the test item was totally rinsed off with no residual test item remained on or in the tissues. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results.

Test Item, Positive Control Item and Negative Control Item

The relative mean viability of the test item treated tissues was 101.7% (>50%) after a 15‑minute exposure period and 42‑hour post‑exposure incubation period.

Traces of residual test item remained on the test item treated tissue culture surfaces after rinsing due to viscosity.

Assessment of Color Interference with the MTT endpoint

The solution containing the test item was a yellow color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

Quality Criteria

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 2.7% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6% (≤18%). The positive control acceptance criteria were therefore satisfied.

The mean OD₅₇₀for the negative control (DPBS) treated tissues was 0.736, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 1.2% (≤18%). The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 14.8% (≤18%). The test item acceptance criterion was therefore satisfied.

Individual and Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of triplicate tissues	±SDof OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item^Å	0.743	0.736	0.009	100.9	100*	1.2
	0.726			98.6
	0.740			100.5
Positive Control Item^Å	0.019	0.020	0.004	2.6	2.7	0.6
	0.016			2.2
	0.024			3.3
Test Item	0.846	0.749	0.109	114.9	101.7	14.8
	0.771			104.7
	0.631			85.7

The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not classified as a skin irritant according to the Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).

Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN^TMreconstructed human epidermis model after a treatment period of 15 minutes followed by a post‑exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3‑[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. 

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT in the pre-test for direct MTT reduction and therefore additional non-viable tissues were incorporated into the testing for correction purposes. At the end of the post exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre labeled micro tubes and stored in a freezer for possible inflammatory mediator IL-1α determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT loaded tissues. 

At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm (OD₅₇₀).

The relative mean viability of the test item treated tissues was 101.7% after the 15‑minute exposure period and 42‑hours post‑exposure incubation period.

The relative mean viability for tissues treated with 5% (w/v) SDS aqueous solution (positive control) was 2.7% relative to the negative control treated tissues and the standard deviation value of the viability was 0.6% (≤18%). The positive control acceptance criteria were therefore satisfied.

The mean OD₅₇₀for the negative control (DPBS) treated tissues was 0.736, within the range of 0.6 - 1.5 and the standard deviation value of the viability was 1.2% (≤18%). The negative control acceptance criteria were therefore satisfied.

The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 14.8% (≤18%). The test item acceptance criterion was therefore satisfied.

The test item was not classified as a skin irritant according to the Regulation (EC) No. 1272/2008 Classification, Labelling and Packaging of Substances and Mixtures (EU CLP).