4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 June 2018 - 13 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.

Test material

Test material information

Constituent 1

Reference substance name:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

EC Number:

276-309-1

EC Name:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

Cas Number:

72058-41-4

Molecular formula:

C11H18Cl2N4

IUPAC Name:

4,6-dichloro-N-(2,4,4-trimethylpentan-2-yl)-1,3,5-triazin-2-amine

Test material form:

solid

Details on test material:

Colour: light yellow

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Suqian Unitech Co., LTD; 2018041002
- Purity: 99.29%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed. The OECD 442C assay was run once, in triplicate.

This in chemico method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its reactivity towards a synthetic cysteine and lysine containing peptide, by measuring the depletion using high performance liquid chromatography (HPLC).

Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following solvents suitable for the test:

- acetonitrile
- dist. water
- dist. water : acetonitrile 1:1 (v/v),

The test item was not soluble in -dist. water, and -dist. water : acetonitrile 1:1 (v/v),. The test item was completely soluble in acetonitrile, therefore, acetonitrile was chosen as suitable vehicle for the main experiments.

Controls
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.

Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.

Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.

Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run (see Appendix 2: Exemplary Analysis Sequence).

Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run (see Appendix 2: Exemplary Analysis Sequence).

Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile RC C for the test item was the same as for the positive control because no (further) solvent was used. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples (see Appendix 2: Exemplary Analysis Sequence).

Peptides
20.00 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.04 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
20.13 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (38.29 mL) to reach a concentration of 0.667 mM.

Dose Groups
Reference Control C (solvent control) undiluted

Test Item 100 mM stock solution

Positive Control 100 mM stock solution


Experimental Procedure
Incubation of the Test Item with the Cysteine and Lysine Peptide
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel. Samples were prepared according to the scheme described in Table 4

Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the following HPLC procedure

Preparation of the HPLC Standard Calibration Curve
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions covering the range indicated in the following table (see Table 5).

HPLC Preparation and Analysis
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days, all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. An example of HPLC analysis sequence is provided in the Appendix 2: Exemplary Analysis Sequence


Data Analysis
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated according to the following formula:


PPD = (1-(peptide peak area in the replicate injection/mean peptide peak area in reference control C))*100

Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. In the framework of an IATA the test substance may be considered as non-sensitiser to skin, if the mean depletion of both peptides is below 6.38%. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive"

Acceptance Criteria
The run meets the acceptance criteria if:

- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Results and discussion

Positive control results:

For the 100 mM stock solution (cysteine peptide), slight precipitation was observed for the samples of the positive control excluding the co-elution control. For the 100 mM stock solution (lysine peptide), phase separation was observed for the samples of the positive control including the co-elution control. Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as not relevant.

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 64.28% (Table 9).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Depletion of the Cysteine Peptide

Parameter:

other: Mean Peptide Depletion [%]

Remarks:

(Test item)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

69.13%

Results 2

Key result

Run / experiment:

other: Depletion of the Lysine Peptide

Parameter:

other: Mean Peptide Depletion [%]

Remarks:

(Test item)

Value:

86.03

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

59.44%

Results 3

Key result

Run / experiment:

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

Parameter:

other: Mean Peptide Depletion [%]

Remarks:

(Test item)

Value:

93.01

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

64.28

Remarks on result:

positive indication of skin sensitisation

Results 4

Run / experiment:

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

Parameter:

other: Mean Peptide Depletion [%]

Remarks:

(Test item)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

69.13

Other effects / acceptance of results:

OTHER EFFECTS:
Pre-Experiments
Solubility of the test item was determined prior to the main experiment. The test item was soluble in acetonitrile. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using acetonitrile. All test item solutions were freshly prepared immediately prior to use.
Precipitation and Phase Separation
All test item solutions were freshly prepared immediately prior to use.

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 observed for the samples of the standard solution (STD1 Cys 0.534), Reference controls (REF A1, REF A2, and REF A3) and for the samples of the positive control excluding the co-elution control. Phase separation was observed for all the samples of the test item. 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, but a turbidity. 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 observed for the samples of the positive control including the co-elution control. The turbidity was gone but precipitation was also observed for all the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.
Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as not relevant.

ACCEPTANCE OF RESULTS:
The controls confirmed the validity of the study for both, the cysteine and lysine run, as shown in Table 10 and Table 11. Historical control data for both peptides were provided (Tables 12, 13).

Any other information on results incl. tables

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model (1)
Mean Cysteine and Lysine PPD	Reactivity Class		DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity		Negative
6.38% < PPD ≤ 22.62%	Low Reactivity		Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.                                          

2DPRA predictions should be considered in the framework of an IATA.                                          

Prediction Model 2

Cysteine 1:10 prediction model (1)
Cysteine PPD	Reactivity Class		DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity		Negative
13.89% < PPD ≤ 23.09%	Low Reactivity		Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

1The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2DPRA predictions should be considered in the framework of an IATA.                                          

Applicant's summary and conclusion

Interpretation of results:

other: As part of a tiered testing strategy, the substance may be considered as a “sensitiser”.

Conclusions:

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed. Under the experimental conditions of this study, the DPRA prediction is considered as positive and the test item 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was considered to have high reactivity towards both peptides.

Executive summary:

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA;183806), 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) in acetonitrile was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 ± 2 h at 25 ± 2.5 °C). Subsequently samples were analysed by HPLC. Reference controls (A, B, C (solvent control)), co-elution controls and a positive control (cinnamic aldehyde in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test.

The acceptance criteria for the calibration curve samples, the reference and co-elution controls, as well as for the study samples were satisfied. The study was therefore considered to be valid. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution Slight precipitation was observed for the samples of the standard solution, reference controls A1, A2, and A3 and for the samples of the positive control excluding the co-elution control. Phase separation was observed for all the samples of the test item. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution, but a turbidity. Phase separation was observed for the samples of the positive control including the co-elution control. The turbidity of the samples was gone but precipitation was also observed for all the samples of the test item  Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as not relevant. 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 64.28%.

A phase separation and a precipitation of the test item with both peptide peaks were observed. However, a positive result can still be used. Sensitising potential 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 (solvent control).  The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (93.01%). Based on the prediction model 1 the test item can be considered as sensitiser.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 429 were also performed. The data generated with this test will be considered in the context of an integrated approache such as IATA, combining the result with other complementary information from the other 2 tests.