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

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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)

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

Test animals / tissue source

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

Test system

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.

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.