1,3,5-tris(3-mercaptopropyl)-1,3,5-triazinane-2,4,6-trione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-01-17 to 2019-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adopted 25th June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitisers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore, the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Test material

Test material information

Constituent 1

Reference substance name:

tris(3-sulfanylpropyl)-1,3,5-triazinane-2,4,6-trione

EC Number:

813-556-1

Cas Number:

78366-85-5

Molecular formula:

C12H21N3O3S3

IUPAC Name:

tris(3-sulfanylpropyl)-1,3,5-triazinane-2,4,6-trione

Test material form:

liquid

Details on test material:

- Name: 1,3,5-Tris-(3-mercaptopropyl)isocyanurate
- CAS: 78366-85-5
- Purity: 94.5% (HPLC)
- Batch No: SG0117
- Molecular weight: 351.5 g/mol
- Physical state: liquid
- Colour: colourless
- Storage conditions: room temperature

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%; AppliChem; Lot No.: 0001336139). A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube. No correction factor will be applied to correct for the purity of the test item on the Sponsor’s request. Vortex mixing was used to aid solubilisation.

Based on the DMSO stock solutions, serial dilutions were made using the solvent (DMSO) to obtain 12 master concentrations of the test item. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2 (for experiments 1-3). In experiment 4 the test item stock solution was diluted by a factor of 1.33. Then the master solutions were further diluted 1:25 in cell culture medium. These 1:25 diluted test item solutions were finally diluted 1:4 in cell culture medium when incubated with the cells. Based on this procedure the final concentration of the solvent (DMSO) was 1% (v/v) in all test item concentrations and controls.

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system)
- Details on study design:

CELL LINE:
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number < 25 were used. Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 ± 1 °C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in test item exposure medium.

LUCIFERASE ASSAY SYSTEM:
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual. The kit (Promega, Cat. No.: E1501, Lot No.: 0000328865, 0000343783) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer.
If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.

Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000265269) consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBG3520)

DOSE GROUPS:
Negative Control: 1% (v/v) DMSO in test item exposure medium
Positive Control: CA: 4 µM, 8 µM, 16 µM, 32 µM, 64 µM
Test Item: 12 concentrations of the test item:
Experiments 1, 2 and 3: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.50 µM, 125.0 µM, 250.0 µM, 500.0 µM, 1000 µM, 2000 µM
Experiment 4: 2.71 µM, 3.61 µM, 4.80 µM, 6.38 µM, 8.49 µM, 11.29 µM, 15.02 µM, 19.97 µM, 26.57 µM, 35.33 µM, 46.99 µM, 62.50 µM
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.

EXPERIMENTAL PROCEDURE:
The incubation was performed in 96-well plates. Cells were counted by Neubauer chamber and a cell suspension of 8 × 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 10^4 cells were dispensed in each well except for the blank. Cells were mixed by swinging during pipetting into the 96-well plate to ensure homogeneous cell number distribution. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the freshly prepared 25 times-diluted master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

LUCIFERASE ACTIVITY:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader (Tecan, Infinite 200Pro) for luminescence measurement. For each well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1000 ms before assessing the luciferase activity for 2000 ms. This procedure was repeated for each individual well of 96-well plate.

CELL VIABILITY:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiments 2 and 3) or over the weekend (experiment 1). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm using a plate reader (Tecan, Infinite 200Pro)

DATA ANALYSIS:
For each test item at least two independent runs using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control.

PREDICTION MODEL:
A KeratinoSens™ prediction is considered positive if the following conditions will be met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p< 0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction

If in a given run, all of the three first conditions are met but a clear dose-response relationship for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations < 1000 µM is considered as inconclusive. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.

Results and discussion

Positive control results:

- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.41 in experiment 1; 3.48 in experiment 2; 2.39 in experiment 3; 3.84 in experiment 4).
- The calculated EC1.5 was between 7 and 34 µM (15.84 µM in experiment 1; 17.73 µM in experiment 2, 28.31 µM in experiment 3, 15.07 µM in experiment 4).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1, at 125 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

3.21

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

the four criteria of the prediction model were not all fulfilled.

Results 2

Key result

Run / experiment:

other: Experiment 2, at 62.50 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

2.9

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

the four criteria of the prediction model were fulfilled

Results 3

Key result

Run / experiment:

other: Experiment 3, 62.50 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

3.13

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

only three of four criteria of the prediction model were fulfilled

Results 4

Key result

Run / experiment:

other: Experiment 4, at 8.49 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.66

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

the four criteria of the prediction model were fulfilled

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The acceptance criteria proposed by the OECD test guideline 442D were met in this test.

For individual results see Table 1 and Table 2 in box 'Any other information on results incl. tables'.

Any other information on results incl. tables

Table 1: Mean luciferase activity induction and cell viability in Experiments 1-3
Experiment 1-3	Concentration [µM]	Fold Induction luciferase activity			Cell Viability [%]
		Exper. 1	Exper. 2	Exper. 3	Exper. 1	Exper. 2	Exper. 3
Solvent Control	-	1.00	1.00	1.00	100	100	100
Positive Control	4.00	1.20	1.18	0.99	88.1	95.7	99.3
	8.00	1.31	1.42	1.08	97.1	93.9	97.3
	16.00	1.50*	1.46	1.20	94.3	95.5	99.9
	32.00	2.13*	1.81*	1.59*	95.0	85.0	87.4
	64.00	4.41*	3.48*	2.39*	94.4	73.7	100.1
Test Item	0.98	1.19	1.25	1.02	105.7	96.0	90.1
	1.95	1.14	1.24	0.97	105.8	108.0	93.8
	3.91	1.49	1.62*	1.11	104.8	114.3	76.3
	7.81	1.45	1.71*	0.94	103.4	101.1	73.9
	15.63	0.84	1.06	0.83	79.1	86.7	66.6
	31.25	1.17	2.13*	1.73*	67.8	66.4	74.2
	62.50	2.55*	2.90*	3.13*	59.7	43.1	25.1
	125.00	3.21*	1.15	0.38	4.8	1.2	0.3
	250.00	0.11	0.01	0.00	0.5	0.3	0.3
	500.00	0.00	0.00	0.00	0.7	0.6	0.7
	1000.00	0.00	0.00	0.00	2.1	2.0	1.8
	2000.00	0.00	0.00	0.00	8.4	9.0	4.3

*= p< 0.05

Table 2: Mean luciferase activity induction and cell viability in Experiment 4
Experiment 4	Concentration [µM]	Fold Induction luciferase activity	Cell Viability [%]
		Exper. 4	Exper. 4
Solvent Control	-	1.00	100
Positive Control	4.00	1.19	101.1
	8.00	1.32	105.6
	16.00	1.52	109.6
	32.00	2.04*	117.0
	64.00	3.84*	88.6
Test Item	2.71	0.92	61.6
	3.61	1.05	62.7
	4.80	1.57*	69.2
	6.38	1.65*	88.9
	8.49	1.66*	52.8
	11.29	1.57*	18.4
	15.02	0.86	0.7
	19.97	0.08	0.2
	26.57	0.01	0.2
	35.33	0.00	0.4
	46.99	0.00	0.5
	62.50	0.00	0.9

*= p< 0.05

Applicant's summary and conclusion

Interpretation of results:

other: positive indication of a sensitising potential

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in two independent experiment runs. Therefore, the test item can be considered to have a sensitising potential.

Executive summary:

In a dermal sensitisation study conducted according to OECD 442D with 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (94.5% purity) in DMSO, the sensitisation potential of the test item was assessed on the basis of the activation of keratinocytes in vitro using the KeratinoSens™ cell line. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity. Sensitisation was scored by measuring maximum luciferase activity induction (I_max), cell viability and EC_1.5. A total of four independent experiments were conducted. Two experiments fulfilled all criteria of the prediction model mentioned in the OECD test guideline 442D, and therefore, these two experiments are considered positive. Based on the results, the test item 1,3,5-Tris-(3-mercaptopropyl)isocyanurate is considered to have a sensitising potential. The data generated with this test should be considered in the context of an integrated approach combining this result with information derived from in vitro assays addressing other key events of the skin sensitisation adverse outcome pathway (AOP).