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

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

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 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

These deviations did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”. Data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of an integrated approach such as IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP

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

Results and discussion

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.21%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Parameter:

other: mean depletion of both peptides

Value:

21.61

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (21.61%). Based on the prediction model 1 the test item can be considered as sensitiser.

Results 2

Key result

Run / experiment:

other: Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)

Parameter:

other: Mean Peptide Depletion of both peptides

Value:

42.91

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Moderate Reactivity

Any other information on results incl. tables

3.1.1.       Results of the Cysteine Peptide Depletion

Table7:  Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1400.8739	0.1518	69.77	70.12	0.39	0.56
	1387.7372	0.1504	70.05
	1364.8569	0.1479	70.54
Test Item	2690.9124	0.2910	42.02	42.91	1.32	3.07
	2678.1470	0.2897	42.29
	2579.2166	0.2790	44.42

 

 

3.1.2.       Results of the Lysine Peptide Depletion

Table8:  Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	2198.9507	0.2087	57.98	58.30	1.46	2.50
	2249.3828	0.2136	57.02
	2099.5659	0.1992	59.88
Test Item	5306.5352	0.5059	0.00	0.31	0.27	87.15
	5269.4561	0.5023	0.44
	5266.2188	0.5020	0.50

3.1.3.       Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Table9:  Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	21.61	Low Reactivity	sensitizer	42.91	Moderate Reactivity	sensitizer
Positive Control	64.21	High Reactivity	sensitizer	70.12	Moderate Reactivity	sensitizer

Thein chemicodirect peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.

In the present study 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione was dissolved in methanol based on the results of the pre-experiments

Based on a molecular weight of 272.44 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight precipitation was noted for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was noted for the samples of the positive control in cluding the co-elution control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitisingpotential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showedlowreactivity towards the synthetic peptides. The mean depletion of both peptides was>6.38% (21.61%). Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.21%.

The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was > 0.99 (0.9999). The mean peptide depletion of the cysteine peptide was between 60.8% and 100% (70.12 %). The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.5128 mM, RC C_acetonitrile: 0.5007 mM, RC C_methanol: 0.5015 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 0.93%, RC C_acetonitrile: 0.84%, RC C_methanol: 0.92%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 14.9% (PC: 0.39%; test item: 1.32%).

For the lysine run the coefficient of determination for the calibration curve was > 0.99 (0.9999). The mean peptide depletion of the lysine peptide was between 40.2% and 69.0% (58.30%).The mean peptide concentration of reference controls A and reference controls C was between 0.45 and 0.55 mM (RC A: 0.4996 mM, RC C_acetonitrile: 0.4989 mM, RC C_methanol: 0.5046 mM). The coefficient of variation of the peak areas of reference controls B and reference controls C was < 15%. (RC B: 0.24%, RC C_acetonitrile: 0.14%, RC C_methanol: 0.43%). The standard deviation of the peptide depletion for the replicates of the positive control as well as for the test item samples was < 11.6% (PC: 1.46%; test item: 0.27%).

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived fromin vitroassays addressing other key events of the skin sensitisation AOP.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed low reactivity towards the peptides. The test item can be classified as “sensitiser” in accordance with UN GHS “Category 1”.
The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study 6-(dibutylamino)-1,3,5-triazine-2,4(1H,3H)-dithione was dissolved in methanol based on the results of the pre-experiments

Based on a molecular weight of 272.44 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution.After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight precipitation was noted for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution.After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was noted for the samples of the positive control including the co-elution control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitisingpotential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C).

The 100 mM stock solution of the test item showedlowreactivity towards the synthetic peptides. The mean depletion of both peptides was >6.38% (21.61%). Based on the prediction model 1 the test item can be considered as sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.21%.