6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

EC Number:

249-682-3

EC Name:

6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

Cas Number:

29529-99-5

Molecular formula:

C11H20N4S2

IUPAC Name:

6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Details on animal used as source of test system:

The test was carried out with the reconstituted three-dimensional human skin model EpiDerm (MatTek). This skin model consists of normal human epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. The NHEK are cultured on chemically modified, collagen-coated cell culture inserts (Millicell). The EpiDerm epidermis model exhibits in vivo-like morphological and growth characteristics which are uniform and highly reproducible. It consists of organised basal, spinous and granular layers and a multi-layered stratum corneum analogous to patterns found in vivo.

Vehicle:

not specified

Control samples:

yes, concurrent negative control

Amount/concentration applied:

25 mg + 25 µL DPBS

Duration of treatment / exposure:

After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue staggered in e.g. one-minute intervals. After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Then all plates were removed from the incubator and placed under the sterile flow until the 60 ± 1 min incubation time of the first dosed tissue was over

Duration of post-treatment incubation (if applicable):

The plates were post-incubated at 37  1 C, 5.0% CO2, humidified to 95%, for 24  2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 96.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerm (MatTek) comprising a reconstructed epidermis with a functional stratum corneum.
In the present study 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione was applied topically to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.
Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.
The test item showed no non-specific MTT-reduction (NSMTT) and no colouring potential, therefore no additional controls were necessary.
The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (96.3%) after 60 min treatment and 42 h post-incubation.
The controls confirmed the validity of the study. The mean absolute OD570 of the three negative control tissues was  0.8 and ≤ 2.8 (1.545). The mean relative tissue viability (% negative control) of the positive control was  20% (3.9%). Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.7% - 11.5%).

Executive summary:

In the present study the skin irritant potential of 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione was analysed.The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404,[7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a60 min exposure and 42 h post-incubation periodand compared to those of the concurrent negative controls.

The test item showed no non-specific MTT-reduction (NSMTT) and no colouring potential, therefore no additional controls were necessary.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 50% (96.3%) after 60 min treatment and 42 h post-incubation.

In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was> 50%. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.