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

Endpoint summary

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 96.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

Approximately 50 mg (83.3 mg/cm2) of the test item was applied directly atop the EpiOcular™ tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue.

Duration of treatment / exposure:

for 6 ± 0.25 h

Duration of post- treatment incubation (in vitro):

18 ± 0.25 h

Number of animals or in vitro replicates:

2

Details on study design:

Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h. Then the inserts were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37  1 °C, 5.0% CO2 / 95% air for 16 - 24 h.
After the overnight incubation, the tissues were pre-treated with 20 µL of DPBS-buffer and incubated for 30 ± 2 min in a humidified incubator at 37  1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37  1 °C, 5.0% CO2 / 95% air. At the end of the exposure period, the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing, the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37  1°C, 5.0% CO2 / 95% air.
After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h  10 min at 37  1 °C, 5.0% CO2 / 95% air.
After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out either after storage overnight in the dark at 2 - 8 C° or immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
Per each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

Irritation parameter:

other: Mean Relative Tissue Viability

Run / experiment:

Main experiment

Value:

ca. 18.7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.441 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 32.7 < 50% pass
Max. Difference of % Viability [%] 1.1 < 20% pass

1.1.1.       Experiment

Table2:  Result of the Test Item 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione

Name	Negative Control		Positive Control		Test Item
Tissue	1	2	1	2	1	2
Aabsolute OD570	1.438	1.432	0.495	0.510	0.312	0.297
	1.449	1.444	0.489	0.504	0.308	0.296
OD570(Blank-Corrected)	1.396	1.390	0.453	0.468	0.270	0.255
	1.407	1.402	0.447	0.462	0.266	0.254
Mean OD570of the Duplicates (Blank-Corrected)	1.401	1.396	0.450	0.465	0.268	0.254
Total Mean OD570of 2 Replicate Tissues (Blank-Corrected)	1.399^*		0.457		0.261
SD OD570	0.01		0.01		0.01
Relative Tissue Viability [%]	100.2	99.8	32.2	33.2	19.2	18.2
Relative Tissue Viability Difference [%]^***	0.4		1.1		1.0
CV [% Viability]	0.4		3.3		5.3
Mean Relative Tissue Viability [%]	100.0		32.7^**		18.7

^*             Corrected mean OD₅₇₀of the negative control corresponds to 100% absolute tissue viability

^**            mean relative tissue viability of the positive control is < 50%

^***           relative tissue viability difference of replicate tissues is < 20%.

1.1.2.       Test Acceptance Criteria

Table3:  Test Acceptance Criteria

	Value	Cut off	pass/fail
Mean Absolute OD570 nmNK	1.441	0.8 < NK < 2.5	pass
Mean Relative Viability PC [%]	32.7	< 50%	pass
Max. Difference of % Viability [%]	1.1	< 20%	pass

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

worst case classification

Conclusions:

In this study under the given conditions the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2

Executive summary:

In the present study theeye irritating potential of 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione was analysed. Since irritant substances are cytotoxic after a short time exposure to the corneal epithelium the cytotoxic effects of the test item on EpiOcularÔ, a reconstituted three-dimensional human corneal epithelium model, were determined. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 30 min exposure period and 18 h post-treatment period and compared to those of the concurrent negative controls.

The test item showed non-specific reduction of MTT but an OD above 0.08 after mixture with isopropanol. In the main experiment, the test item showed irritant effects. The mean relative tissue viability (% negative control) was reduced below 60% (18.7%). Therefore, no additional controls for correction of results were necessary.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin irritation:

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”

Eye irritation:

In the studies under the given conditions the test item showed irritant effects.

No prediction can be made regarding corrosivity of the test substance 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione according to the evaluation criteria. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2

Therefore the substance is "worst-case" classified as “corrosive” in accordance with UN GHS “Category 1”