1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 December 2016 - 15 May 2017

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol no 154,

Version / remarks:

2013

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay

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 adverse outcome pathway.

Test material

Test material information

Constituent 1

Reference substance name:

1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

EC Number:

222-148-7

EC Name:

1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

Cas Number:

3371-33-3

Molecular formula:

C17H19NO4.BrH

IUPAC Name:

1-(3,5-dihydroxyphenyl)-2-{[1-(4-hydroxyphenyl)propan-2-yl]amino}ethan-1-one hydrobromide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:TH 1165 Ketone II BR (Batch No: 159)
- Expiration date of the lot/batch: 30 April 2017
- Purity test date:No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions:stable at storgae conditions
- Solubility and stability of the test substance in the solvent/vehicle: The test item was prepared immediatlely prior to use.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No details reported.
- Preliminary purification step (if any):No details reported.
- Final dilution of a dissolved solid, stock liquid or gel:No details reported.
- Final preparation of a solid:No details reported.

In chemico test system

Details on the study design:

- Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. 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.

- 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 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.
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.
Reference control C was set up for the test item and the positive control. Reference control C for the test item was prepared using the respective solvent used to solubilize the test item. RC C for the positive control was prepared using acetonitrile. Reference control 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. Reference control C was included in every assay run for both peptides and was injected together with the samples.

- Test System
For HPLC system and HPLC mobile phase see table 1 and 2.

- Peptides
15.20 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (29.52 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
19.44 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 (36.32 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

- Dose Groups
Reference Control C (solvent control): undiluted
Test Item: 100 mM stock solution
Positive control: 100 mM stock solution

- Pre-Experiments
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) The test item was dissolved in the following solvents suitable for the test:
acetonitrile
dist. water
isopropanol
acetone
DMSO : acetonitrile 1:10 (v/v)
DMSO : acetonitrile 1:10 (v/v)
Methanol
Since the test item gave a stable solution in Methanol, Methanol was chosen as suitable vehicle.

- 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 3.

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 HPLC.

- Data Analysis:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at lambda = 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 in accordance with UN GHS “Category 1”, 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. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) shown in table 4 the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin in accordance with UN GHS “No Category” if the mean depletion of both peptides is below 6.38% (see Table 4 and 5).

- 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:

The reactivity of the positive control towards the cysteine peptide and peptide were identified correctly and both runs were valid.

In vitro / in chemico

Results

Key result

Run / experiment:

other: Cysteine Peptide Depletion

Parameter:

other: % mean peptide depletion

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Pre-Experiments:
Since the test item gave a stable solution in Methanol, Methanol was chosen as suitable vehicle.

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.
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 of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the positive control and the test item. Samples were not centrifuged prior to the HPLC analysis.
After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. Turbidity and phase separation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis.

Co-elution with the Peptide Peaks:
Co-elution of the test item with the lysine peptide peak was observed. Therefore, data were interpreted using Prediction Model 2.

Results Calibration Curve
Cysteine Peptide Calibration Curve: y = 8439.71x-31.13; R² = 0.9997
Lysine Peptide Calibration Curve: y = 8464.89x+4.68; R² = 0.9999

For detailed results see table 6 to10.

Any other information on results incl. tables

Table 6: Results of the Cysteine Peptide Depletion

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	906.9033	0.1111	78.63	78.90	0.58	0.74
	867.1926	0.1064	79.57
	912.4229	0.1118	78.50
Test Item	0.0000	0.0037	100.00	100.00	0.00	0.00
	0.0000	0.0037	100.00
	0.0000	0.0037	100.00

 Since the co-elution control (only containing test item and solvent) showed a peak at the retention time of the cysteine peptide with a peak area of 284.9609, the peak areas detected for the peptide in the test item samples might not (only) represent the remaining cysteine peptide. Therefore, the true peptide depletion might be higher and underestimation of the sensitizing potential is possible.

 

Table 7: Results of the Lysine Peptide Depletion

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	2051.3940	0.2418	52.31	51.62	0.72	1.39
	2078.4993	0.2450	51.68
	2113.1423	0.2491	50.87
Test Item	2107.8921*	n.a.	n.a.	n.a.	n.a.	n.a.
	2159.4229*	n.a.	n.a.
	2098.2346*	n.a.	n.a.

* Since co-elution of the test substance with the lysine peptide was observed, the given peak areas of the lysine peptide in combination with the test item are considered as insignificant. Therefore, they were not used for calculation or evaluation.

Table 8: Categorization of the Test Item

Predicition 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	n.a.*	n.a.*	n.a.*	100.00	High Reactivity	sensitizer
Positive Control	65.26	High Reactivity	sensitizer	78.90	Moderate Reactivity	sensitizer

* Not applicable due to co-elution of the test substance with the lysine peptide

 

Table 9: Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9997	pass
mean peptide concentration of RC A	0.45 = x = 0.55 mM	0.5056	pass
mean peptide concentration of RC C (PC)	0.45 = x = 0.55 mM	0.5066	pass
mean peptide concentration of RC C (TI)	0.45 = x = 0.55 mM	0.5003	pass
CV of the peak area of RC B	< 15%	1.29	pass
CV of the peak area of RC C (PC)	< 15%	0.59	pass
CV of the peak area of RC C (TI)	< 15%	0.59	pass
mean peptide depletion of the PC	60.8% < x < 100%	78.90	pass
SD of peptide depletion of the PC replicates	< 14.9%	0.58	pass
SD of peptide depletion of the TI replicates	< 14.9%	1.05	pass

 

Table 10: Acceptance Criteria for Lysine Peptide

Lysine Peptide Run
Acceptance Criterion	Range	Value	pass/fail
coefficient of determination	R² > 0.99	0.9999	pass
mean peptide concentration of RC A	0.45 = x = 0.55 mM	0.5075	pass
mean peptide concentration of RC C (PC)	0.45 = x = 0.55 mM	0.5076	pass
mean peptide concentration of RC C (TI)	0.45 = x = 0.55 mM	0.5073	pass
CV of the peak area of RC B	< 15%	0.23	pass
CV of the peak area of RC C (PC)	< 15%	0.18	pass
CV of the peak area of RC C (TI)	< 15%	0.52	pass
mean peptide depletion of the PC	40.2% < x < 69.0%	51.62	pass
SD of peptide depletion of the PC replicates	< 11.6%	0.72	pass
SD of peptide depletion of the TI replicates	< 11.6%	n.a.*	pass

* Not applicable due to co-elution of the test substance with the lysine peptide.

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 moderate reactivity towards the cysteine peptide. Based on the results of this study, the test item can be classified as a “sensitiser” in accordance with UN GHS “Category 1”.

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 the test item was dissolved in Methanol. Based on a molecular weight of 382.26 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.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the positive control. No centrifugation was necessary to perform the HPLC analysis.

After the HPLC run samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. Turbidity and phase separation was observed for the samples of the positive control. No centrifugation was necessary to perform the HPLC analysis.

Since the reactivity of the positive control towards the cysteine peptide and peptide were identified correctly and both runs were valid the precipitation and phase separation was considered as irrelevant.

Co-elution of test itemwith both peptide peaks wasobserved, therefore prediction model 2 is used for data interpretation. However, evaluation of the cysteine peptide was possible, since the peak at the retention time of the peptide but resulting from the test item would not have overlaid the peptide peak regarding peak area and height. Therefore, sensitisingpotential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C.

The 100 mM stock solution of the test item showed high reactivity towards the cysteine peptide, the mean depletion was > 13.89% (100%). Based on the prediction model 2 the test item is considered as a "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 65.26% (Positive Sensitiser DPRA Prediction, Moderate Reactivity; Prediction Model 2).

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 from in vitro assays addressing other key events of the skin sensitisation AOP.