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

Endpoint summary

Description of key information

The potential of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (target substance) to induce skin sensitisation was evaluated in two suitable in vitro test methods conducted according to OECD 442D and OECD 442E. Based on the results, the target substance can be considered as skin sensitiser. Therefore, classification as Skin Sens. 1, H317 is warranted in accordance with the CLP regulation 1272/2008.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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.

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.

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).

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.

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

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

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'.

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

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).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-01-16 to 2019-04-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The correlation of upregulation of immunologically relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event of the skin sensitisation process as described by the AOP for skin sensitisation. This method, which measures the markers of DC activation, using the DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was freshly prepared immediately prior to use. The test item was soluble in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL. Vortex mixing, sonication and warming to 37 °C were used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. Precipitates were observed in the dose finding assay when diluted 1:250 in cell culture medium. Sonication was used to aid solubilisation. A slight turbidity was observed in the main experiment 2 when diluted 1:250 in cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (DMSO) was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Details on the study design:

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

CELL LINE:
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10^6 cells/mL.
Cells were cultured in 75 cm² culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/ 100 µg/mL streptomycin at 37 +/- 1 °C and 5% CO2.

PRE-EXPERIMENTS:
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards defined controls. Hereby, DNCB at a final concentration of 4 µg/mL and nickel sulphate (NiSO4) at a final concentration of 100 µg/mL served as positive control while lactic acid (LA) at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

SOLVENT FINDING:
Solubility of the test item was determined prior to the main experiment. The test item was dissolved in 0.9% NaCl at a final concentration of 500 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 2 down to a minimal concentration of 1 mg/mL.

EXPERIMENTAL PROCEDURE:
Dose Finding Assay
Starting from 500 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding appropriate solvent (DMSO). These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 µL of the cell suspension were seeded into onto a 24 well flat-bottom plate (1 x 10^6 cells/well). The solvent controls and the test item working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer. 200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL. The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration. The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 EXPRESSION
The test item was dissolved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.2^2; CV75/1.2^3; CV75/1.2^4; CV75/1.2^5; CV75/1.2^6. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, FITC-labelled anti-CD54, or FITC-labelled mouse IgG1 (isotype) antibodies in the dark for 30 min at 4 °C. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL). The expression levels of CD86 and CD54 as well as cell viability (as determined by living cells with no PI uptake) were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was also calculated.

DATA ANALYSIS:
FACS data analysis was performed using the software BD FACS DIVA 6.0. Further data analysis like calculation of the CV75, calculation of the RFI and calculation of the Effective Concentration 150 (EC150) and Effective Concentration 200 (EC200) values were performed using the software Microsoft Excel 2010. The mean values and standard deviations of the single replicates were determined using the respective excel commands.

EVALUATION of RESULTS:
For CD86/CD54 expression measurement, the test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided, that for each run, independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used. Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT is considered positive if any of the three scenarios are met:
- the RFI of CD86 is equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs;
- the RFI of CD54 is equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent run;
- the RFIs of both the CD86 and CD54 are equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs.
In case of non-concordant results, a third run should be conducted to make the final prediction. Otherwise the result was considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is < 90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities > 90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (i.e. 5000 µg/mL for 0.9% NaCl; 1000 µg/mL for DMSO or another organic solvent) even if the cell viability is > 90%. A negative result for test items with a Log KOW > 3.5 should be considered as inconclusive.

Positive control results:

The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (289% experiment 1; 305% experiment 2) and 200% for CD54 (284% experiment 1; 255% experiment 2) were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: highest CD86 upregulation

Remarks:

at 8.35 µg/mL

Value:

313

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: highest CD54 upregulation

Remarks:

at 8.35 µg/mL

Value:

1 567

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: highest CD86 upregulation

Remarks:

at 8.35 µg/mL

Value:

159

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: highest CD54 upregulation

Remarks:

at 8.35 µg/mL

Value:

350

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: see Table 3 in box "Any other information on results incl. tables".

For individual results see Tables 1 and 2 in box "Any other information on results incl tables"

Summary of results:

In the present study 1,3,5-Tris-(3-mercaptopropyl)isocyanurate was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 3.91 mg/mL to 500 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 20.77 ± 0.96 µg/mL was derived in the dose finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 24.92, 20.77, 17.31, 14.42, 12.02, 10.02, 8.35 and 6.96 µg/mL. In the dose finding assay precipitation and in the main experiment 2 slight turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium. Cells were incubated with the test item for 24 h at 37 °C. After exposure, cells were labelled with CD54 and CD86 fluorescent antibodies and the expression levels of CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

Cytotoxicity was observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 65.1% (CD86), 64.8% (CD54) and 65.0% (isotype IgG1 control) compared to the solvent control in the first experiment and to 64.4% (CD86), 66.0% (CD54) and 63.9% (isotype IgG1 control) compared to solvent control in the second experiment. In the first experiment the expression of the cell surface marker CD86 was upregulated to a maximum of 313% (8.35 µg/mL) relative to the solvent control. An upregulation above the threshold of 150% was observed at all concentration steps. The expression of the cell surface marker CD54 was upregulated to a maximum of 1567% (8.35 µg/mL) relative to the solvent control. An upregulation above the threshold of 200% was observed at all concentration steps.

In the second experiment the expression of the cell surface marker CD86 was upregulated to a maximum of 159% relative to the solvent control. An upregulation above the threshold of 150% was observed only at a concentration of 8.35 µg/mL. The expression of the cell surface marker CD54 was upregulated to a maximum of 350% (8.35 µg/mL) relative to the solvent control. An upregulation above the threshold of 200% was observed at all concentration steps.

Since the expression of both cell surface markers clearly exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Table 1: CD54 and CD86 Expression Experiment 1

Sample	Conc. μg/mL	Cell Viability [%]			Mean Fluorescence Intensity			Corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.3	96.6	96.6	2482	976	580	1902	396	120	89	428	168
Solvent Control	0.20%	97.2	97.2	96.6	2151	1012	566	1585	446	100	100	380	179
DNCB	4.00	88.7	89.7	88.6	5213	1903	637	4576	1266	289	284	818	299
Test item	8.35	89.9	89.8	89.6	5625	7655	664	4961	6991	313	1567	847	1153

 

Table 2: CD54 and CD86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			Corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI) [%]		Ratio Isotype IgG1 to [%]
		CD86	CD54	IgG Isotype	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	94.4	93.9	94.2	3881	1821	681	3200	1140	116	187	570	267
Solvent Control	0.20%	94.2	94.6	94.8	3420	1269	659	2761	610	100	100	519	193
DNCB	4.0	89.1	89.2	89.0	9098	2245	688	8410	1557	305	255	1322	326
Test item	8.35	76.6	76.5	76.5	5045	2791	657	4388	2134	159	350	768	425

 

Table 3: Historical data

Criterion	mean	SD	N
cell viability solvent controls [%]	97.0	1.3	672
number of test doses with viability > 50%	-	-	1786
RFI of positive control of CD86	401.0	146.8	112
RFI of positive control of CD54	576.6	312.0	112
RFI of solvent control of CD86	115.0	15.1	112
RFI of solvent control of CD54	118.8	25.5	112
MFI ratio IgG1/CD86 for medium control [%]	202.4	50.0	112
MFI ratio IgG1/CD86 for DMSO control [%]	221.6	58.5	112
MFI ratio IgG1/CD54 for medium control [%]	141.0	24.7	112
MFI ratio IgG1/CD54 for DMSO control [%]	147.7	25.6	112

Interpretation of results:

other: positive indication of a sensitising potential

Conclusions:

In this study under the given conditions the test item did trigger an upregulation of the expression of the cell surface markers CD54 and CD86 in two independent experimental runs. Based on these results, the test item can be considered to have a sensitising potential.

Executive summary:

In a skin sensitisation study conducted according to OECD 442E with 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (94.5% purity), the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells in vitro using the human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers.

Sensitisation was scored by measuring cell viability and checking the expression of both cell surface markers. The expression of the cell surface marker CD86 was upregulated above the threshold of 150% at all tested concentrations in the first experiment and at a concentration of 8.35 µg/mL (159%) in the second experiment. The expression of the cell surface marker CD54 was upregulated above the threshold of 200% at all tested concentrations in the first and second experiment. Since the expression of both cell surface markers clearly exceeded the threshold in two independent experiments the test item can be 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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The potential of 1,3,5-Tris-(3-mercaptopropyl)isocyanurate (target substance) to induce skin sensitisation was evaluated in two suitable in vitro test methods conducted according to OECD 442D and OECD 442E. In the first study conducted according to OECD 442D, 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.

In the second study, conducted according to OECD 442E, the sensitisation potential of the test item was assessed on the basis of the activation of dendritic cells in vitro using the human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers. In both studies, the target substance was tested positive and can be considered to have a sensitising potential. Therefore, based on the results, classification as Skin Sens. 1, H317 is warranted in accordance with the CLP regulation 1272/2008.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In two in vitro skin sensitisation studies conducted according to OECD 442D and OECD 442E, the target substance was tested positive for skin sensitisation. Based on the results, classification for skin sensitisation is warranted (Skin Sens. 1, H317).