Carbamic acid, N-[(butylthio)thioxomethyl]-, butyl ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 July 2013 - 02 September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Also in accordance with GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2012)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2010)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

EC Number:

691-061-8

Cas Number:

1001320-38-2

Molecular formula:

C10H19NO2S2

IUPAC Name:

butyl N-[(butylsulfanyl)methanethioyl]carbamate

Test material form:

solid

Remarks:

yellow grease-like solid

Specific details on test material used for the study:

Molecular formula: C10H19NO2S2
Name: n-butoxycarbonyl n-butyl dithiocarbamate (active ingredient)
CAS Number: 1001320-38-2

In vitro test system

Test system:

human skin model

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN small model, 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.
- Tissue batch number(s): Lot no.: 13-EKIN-025 and 13-EKIN-029
- Delivery date: 13-EKIN-014, received: 16 April 2013 and 13-EKIN-019, received: 22 May 2013
- Date of initiation of testing:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 15 minutes after exposure, phosphate buffered saline

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium. Further use of killed tissues was similar to living tissues.
- N. of replicates : 3
- Method of calculation used:

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42h of post incubation is less than or equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42h of post incubation is greater than 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amounts applied: The test substance was melted and the liquid test substance was applied undiluted (25 μl) directly on top of the tissue.

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

56

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

12

Other effects / acceptance of results:

* Negative and positive controls were within historical data range.
* The SD of the positive control was 23% in the first experiment and therefore a repeat experiment was performed.
*The non-specific reduction of MTT by the test material was 8% of the negative control tissues in the first experiment and 22% in the repeat experiment.Since the test substance was difficult to remove, a high non specific MTT resulted in a low viability of the test substance treated tissues.

Since there was a high variation in tissue viability between all individual treated tissues (first experiment: 57, 55 and 57%, repeat experiment: 14, 6 and 17%), no consistent outcome was observed in the six individual treated tissues, and no conclusion can be drawn from this assay.

Applicant's summary and conclusion

Interpretation of results:

other: inconclusive

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. Since there was a high variation in tissue viability between all individual treated tissues (first experiment: 57, 55 and 57%, repeat experiment: 14, 6 and 17%), no consistent outcome was observed in the six individual treated tissues, and thus no conclusion can be drawn from this assay.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (undiluted 25 μl). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT.

Since the test substance was difficult to remove, a high non specific MTT resulted in a low viability of the test substance treated tissues.

Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 18% and 23% in the first and repeat experiment respectively. Whereas the test substance showed cell viability of 56% and 12% in the first and repeat experiment respectively. Since there was a high variation in tissue viability between all individual treated tissues (first experiment: 57, 55 and 57%, repeat experiment: 14, 6 and 17%), no consistent outcome was observed in the six individual treated tissues, no conclusion can be drawn from this assay.