Registration Dossier

Administrative data

Description of key information

Skin sensitisation (in vitro):

1. Direct Peptide Reactivity Assay (DPRA): Positive (OECD 442C/GLP)

2.ARE-Nrf2 luciferase test assay: Positive (OECD 442D/GLP)

The results of the in chemico and in vitro tests were both positve so OECD 442E was not performed; however the results did not allow discrimmination between Skin sensitiser category 1A and 1B. There were no potential source candidates found for read-across so, as a last resort, in vivo testing (OECD 429) was performed.

Skin sensitisation (in vivo): Skin sensitiser Category 1 (OECD429, LLNA/GLP)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
07 December 2018 - 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
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 test date: 99.29%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:

FORM AS APPLIED IN THE TEST
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.: 0001179895; 0001336139). A stock solution of 200 mM was prepared by preweighing the test material into a glass vial. Vortex mixing was used to aid solubilisation. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The animals were derived from a controlled full-barrier maintained breeding system (SPF).
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 17.6-20.4 g
- Housing: Full barrier in an air-conditioned room; the animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding.
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at last 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark
Vehicle:
dimethyl sulphoxide
Concentration:
Pre-screen test: 25% and 50% (w/v)
Main study: 0, 6.25%, 12.5% and 25% (w/v)
No. of animals per dose:
Pre-screen test: 2 mice per dose
Main study: 5 mice per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The maximum technically applicable concentration of the test item in the vehicle was found to be 50% (w/v) in DMSO. Two animals were treated with a test item concentration of 50% (diluted with DMSO). Two animals were treated with a test item concentration of 25% (diluted with DMSO).

- Irritation: Sticky fur was observed at both application sites of each animal treated with the test item from day 3 until day 5. Furthermore, it was noted that the test item at the concentrations of 25% and 50% in DMSO had a very intensive smell.

- Systemic toxicity: Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge). The animals treated with the test item at a concentration of 50% in DMSO showed moderately reduced spontaneous activity on day 2 and slightly reduced spontaneous activity on day 3. The animals treated with the test item at a concentration of 25% in DMSO showed slightly reduced spontaneous activity on day 2 and 3. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the duration of the prescreen test.

- Ear thickness measurements: Ear thickness increased in all dose groups at Day 6

The test item concentration 25% in DMSO was selected as the maximum dose level to use in the main test. According to OECD guideline 429, marked change in activity level might indicate systemic toxicity. Therefore, the test item concentration 50% in DMSO was excluded as both treated animals showed moderately reduced spontaneous activity during the prescreen test.

Refer to Tables 2-4.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: 1% phenylenediamine; a shared positive control was performed concomitantly, using 5 animals.
Positive control results:
The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system (10.4 ± 2; Table 7).
Parameter:
SI
Value:
14
Test group / Remarks:
6.25%
Remarks on result:
not determinable
Parameter:
SI
Value:
18.8
Test group / Remarks:
12.5%
Remarks on result:
not determinable
Parameter:
SI
Value:
12.6
Test group / Remarks:
25%
Remarks on result:
not determinable
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA/DETAILS ON STIMULATION INDEX CALCULATION/EC3 CALCULATION

All of the three tested concentrations of the test item exceeded the stimulation index of 3. The stimulation index at a concentration of 6.25% was 14.0 The stimulation index at a concentration of 12.5% was 18.8 The stimulation index at a concentration of 25% was 12.6 (Table 5). As an excessive local skin irritation by the test item cannot be excluded (see clinical observations below), the data were considered as not conclusive.

CLINICAL OBSERVATIONS: All animals survived throughout the test period without showing any systemic effects. However, significant local effects were recorded on day 6 in all animals treated with the test item. At both application sites signs of alopecia, hardening of the ears, oedema and crust were observed. During excision of the draining auricular lymph nodes it was noted that the ear tissue of all animals treated with the test item at a concentration of 12.5% and 25% was necrotic, detached from surrounding skin tissue and undermined with pus-like discharge (Table 8)

BODY WEIGHTS: All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study (Table 6).

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the conditions of the present study the sensitising properties of the test item 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine could not be fully determined. With the significant local effects observed, a relevant impact on the test results cannot be excluded. Therefore, the results obtained are considered as inconclusive and further testing is required.
Executive summary:

In a dermal sensitization study (188590) with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%), young adult CBA/Ca/Ola/Hsd mice (5 females) were tested in the Local Lymph Node Assay. The doses tested were 0, 6.25, 12.5 and 25% (w/v) in DMSO, based on the results of the pre-screen test (25 and 50% (w/v)).The reliability of the test system was confirmed by a positive control test with 1% phenylenediamine in DMSO that was performed concomitantly, using 5 animals.

The positive control, 1% phenylenediamine, gave the appropriate response (stimulation index 10.4 ± 2). All animals survived throughout the test period without showing any systemic effects. However, significant local effects were recorded on day 6 in all animals treated with the test item. At both application sites signs of alopecia, hardening of the ears, oedema and crust were observed. During excision of the draining auricular lymph nodes it was noted that the ear tissue of all animals treated with the test item at a concentration of 12.5% and 25% was necrotic, detached from surrounding skin tissue and undermined with pus-like discharge. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study. Treatment with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine at 6.25, 12.5 and 25% (w/v) in DMSO resulted in stimulation indices of 14, 18.8 and 12.6, respectively. As an excessive local skin irritation by the test item cannot be excluded, an EC3 value could not be derived and the data were considered as not conclusive; further testing is required.

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
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
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.
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.


Details on 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.


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).
Key result
Parameter:
other: Mean Peptide Depletion [%]
Remarks:
(Test item)
Run / experiment:
Depletion of the Cysteine Peptide
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
69.13%
Key result
Parameter:
other: Mean Peptide Depletion [%]
Remarks:
(Test item)
Run / experiment:
Depletion of the Lysine Peptide
Value:
86.03
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks:
59.44%
Key result
Parameter:
other: Mean Peptide Depletion [%]
Remarks:
(Test item)
Run / experiment:
Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)
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
Parameter:
other: Mean Peptide Depletion [%]
Remarks:
(Test item)
Run / experiment:
Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
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).

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.                                          

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.


Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 June 2018 - 22 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
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 KeratinoSens™ assay showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing.
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; 72058-41-4
- Purity: 99.29%

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- 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.: 0001179895; 0001336139). A stock solution of 200 mM was prepared by preweighing the test material into a glass vial. Vortex mixing was used to aid solubilisation.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

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

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C and OECD 429 were also performed.

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 (P 08 in experiment 1; P 11 in experiment 2) 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. Cells were maintained/assay in DMEM + with 1.0 g/L D-glucose, 1 mM Na-Pyruvate + 10% FCS and tested in DMEM + with 1.0 g/L D-glucose, 1 mM Na-Pyruvate + 1% FCS.

Luciferase Assay System
The luciferase activity was determined using products purchased from Promega. All components were used according to the instructions of the manufacture manual.

Control Groups:
1. Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
2. Negative Control
DMSO (AppliChem; Lot No.: 0001179895; 0001336139) at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
3. Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%; Alfa Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0001179895; 0001336139) at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Dose Groups:
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
3. Test Item: 12 concentrations of the test item
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:
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. 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 shortly before prepared 4x 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 crosscontamination 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 passive 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 for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution (stock solution: 5 mg/mL MTT in DPBS) 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 (experiment 1) or over the weekend (experiment 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Data Analysis
For each test item two independent repetitions 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. In case of discordant results a third independent run is performed.
The following parameters were calculated:Cell Viability, Maximal Induction of the Luciferase Activity (Imax), EC1.5, IC50 and IC30

Positive control results:
The gene induction in the positive control, Cinnamic aldehyde, was above the threshold of 1.5 in 2 of the 5 tested concentrations in run 1 and 2 of the 5 concentrations in run 2.

(Tables 2,3)
Parameter:
other: Imax
Run / experiment:
1
Value:
12.35
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: 86.5% viabilty; 7.81 μM
Parameter:
other: Imax
Run / experiment:
2
Value:
36.74
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: 44.4% viability; 7.81 μM
Parameter:
other: Imax
Run / experiment:
mean
Value:
24.55
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Imax SD = 17.24; IC30 = 7.55 [μM]; IC50 = 9.25 [μM]
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition (Run1: 11.7; Run 2; 8.5).
- Acceptance criteria met for positive control: The gene induction in the positive control, Cinnamic aldehyde, was above the threshold of 1.5 in 2 of the 5 tested concentrations in run 1 and 2 of the 5 concentrations in run 2. The average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8 (Run 1:4.45 and Run 2: 3.62). The EC1.5 value of the positive control is within two standard deviations of the historical mean (Run 1:17.70 and Run 2: 17.02 compared to historical mean 18.5 (n=96)) (Tables 2,3,6,7)
- Range of historical values if different from the ones specified in the test guideline: Historical control data from 2015-2018 was provided (n=96) (Table 7)
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 442C and OECD 429 were also performed. Under the experimental conditions of this study, the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as sensitiser.
Executive summary:

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay; (183807), 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (in 1% DMSO in DMEM culture medium) was applied to KeratinoSens cells at concentrations from 0.98 to 2000 µM for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. The positive control was Cinnamic aldehyde.

In the first experiment, a max luciferase activity (Imax) induction of 12.35 was determined at a test item concentration of 7.81 μM. The corresponding cell viability was 86.5%. The lowest tested concentration with a significant luciferase induction >1.5 (2.20) was found to be 3.91 μM. The corresponding cell viability was >70% (96.9%). The calculated EC1.5 was <1000 μM (2.45 μM).

In the second experiment, a max luciferase activity (Imax) induction of 36.74 was determined at a test item concentration of 7.81 μM. The corresponding cell viability was 44.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.62) was found to be 0.98 μM. The corresponding cell viability was >70% (101.9%). No EC1.5 value could be calculated. The mean Imax for both experiments was 24.55 ± 17.24. A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. The controls confirmed the validity of the study.  Under the condition of this study the test item is therefore considered as sensitiser.

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation (in vitro):

Two in vitro skin sensitisation assays are available: Direct Peptide Reactivity Assay (DPRA) and ARE-Nrf2 luciferase test assay.

Direct Peptide Reactivity Assay (DPRA)

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA;OECD 442C/GLP), 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.

ARE-Nrf2 luciferase test assay

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay (OECD 442D/GLP), 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (in 1% DMSO in DMEM culture medium) was applied to KeratinoSens cells at concentrations from 0.98 to 2000 µM for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. The positive control was Cinnamic aldehyde. In the first experiment, a max luciferase activity (Imax) induction of 12.35 was determined at a test item concentration of 7.81 μM. The corresponding cell viability was 86.5%. The lowest tested concentration with a significant luciferase induction >1.5 (2.20) was found to be 3.91 μM. The corresponding cell viability was >70% (96.9%). The calculated EC1.5 was <1000 μM (2.45 μM). In the second experiment, a max luciferase activity (Imax) induction of 36.74 was determined at a test item concentration of 7.81 μM. The corresponding cell viability was 44.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.62) was found to be 0.98 μM. The corresponding cell viability was >70% (101.9%). No EC1.5 value could be calculated. The mean Imax for both experiments was 24.55 ± 17.24. A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. The controls confirmed the validity of the study.  Under the condition of this study the test item is therefore considered as sensitiser.

The results of the in chemico and in vitro tests were both positve so OECD 442E was not performed; however the results did not allow discrimmination between Skin sensitiser category 1A and 1B. There were no potential source candidates found for read-across so, as a last resort, in vivo testing (OECD 429) was performed.

Skin sensitisation (in vivo):

There is one in vivo test (LLNA) available in mice.

In a dermal sensitization study (OECD 429/GLP) with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%), young adult CBA/Ca/Ola/Hsd mice (5 females) were tested in the Local Lymph Node Assay. The doses tested were 0, 6.25, 12.5 and 25% (w/v) in DMSO, based on the results of the pre-screen test (25 and 50% (w/v)).The reliability of the test system was confirmed by a positive control test with 1% phenylenediamine in DMSO that was performed concomitantly, using 5 animals. The positive control, 1% phenylenediamine, gave the appropriate response (stimulation index 10.4 ± 2). All animals survived throughout the test period without showing any systemic effects. However, significant local effects were recorded on day 6 in all animals treated with the test item. At both application sites signs of alopecia, hardening of the ears, oedema and crust were observed. During excision of the draining auricular lymph nodes it was noted that the ear tissue of all animals treated with the test item at a concentration of 12.5% and 25% was necrotic, detached from surrounding skin tissue and undermined with pus-like discharge. All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study. Treatment with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine at 6.25, 12.5 and 25% (w/v) in DMSO resulted in stimulation indices of 14, 18.8 and 12.6, respectively. As excessive local skin irritation by the test item cannot be excluded, an EC3 value could not be derived and the data were considered as not conclusive; further testing is required.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information in the dossier, the substance 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (CAS No. 72058-41-4) is classified as a skin sensitiser 1 when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied.