4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-01 to 2018-01-20

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February 04, 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Cas Number:

1115233-52-7

Molecular formula:

C21 H18 Cl F

IUPAC Name:

4-chloromethyl-4''-ethyl-2'-fluoro-[1,1':4',1''-terphenyl]

Test material form:

solid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: 20.32 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (39.48 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM. 20.56 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 (39.09 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.
- Preparation of the test chemical solutions: The test item was freshly prepared immediately prior to use. 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 a pre-experiment the test item was not soluble in acetonitrile, dist. water, dist. water : acetonitrile 1:1 (v/v), isopropanol, methanol, 1,4-butanediol. The test item was completely soluble in N,N-dimethylformamide(DMF), therefore, DMF was chosen as suitable vehicle for the main experiments.
- Preparation of the positive controls, reference controls and co-elution controls:
Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100mM 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 220nm 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. 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. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. 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.

INCUBATION
- Incubation conditions: 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 1 (see “Any other information on materials and methods”).
- Precipitation noted:
For the 100 mM solution of the test item 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. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item 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. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: 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 in the range from 0.0 to 0.534 mM.
- Verification of the suitability of the HPLC for test chemical and control substances: 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. For details on HPLC system refer to “Any other information on materials and methods”.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: λ = 220 nm

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 62.40 %.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

5.58

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.21

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

other: Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: not applicable

DEMONSTRATION OF TECHNICAL PROFICIENCY: not specified

ACCEPTANCE OF RESULTS:
Please refer to table 8 and 9 under “Any other information on results”.

Any other information on results incl. tables

 

Table 2: Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	5221.6792	0.5340	4254.6284	0.5340
STD2	2648.5273	0.2670	2161.7087	0.2670
STD3	1327.5009	0.1335	1051.8925	0.1335
STD4	670.7400	0.0667	525.2503	0.0667
STD5	334.8562	0.0334	260.5259	0.0334
STD6	166.3505	0.0167	130.8325	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

 

Table 3: 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	1609.4324	0.1633	68.11	68.17	0.05	0.08
	1604.4860	0.1628	68.21
	1605.4248	0.1629	68.19
Test Item	4722.0444	0.4814	4.97	5.58	0.54	9.77
	4682.2471	0.4774	5.77
	4670.3384	0.4761	6.01

 

Table 4: 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	1675.9452	0.2100	57.93	56.62	1.13	2.00
	1753.5808	0.2197	55.98
	1754.5223	0.2198	55.95
Test Item	4024.7078	0.5039	0.13	0.21	0.15	72.44
	4025.2893	0.5039	0.12
	4014.3962	0.5026	0.39

 

Table 5: Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine 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

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

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

 

 

Table 6: Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	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

 

Table 7: 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	-	-	-	5.58	Minimal Reactivity	negative
Positive Control	62.40	High Reactivity	positive	68.17	Moderate Reactivity	positive

 

Table 8: Acceptance Criteria for Cysteine Peptide

Cysteine 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	0.5193	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.5147	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.5066	pass
CV of the peak area of RC B	<15 %	0.44	pass
CV of the peak area of RC C (PC)	<15 %	0.53	pass
CV of the peak area of RC C (TI)	<15 %	0.34	pass
mean peptide depletion of the PC	60.8 % < x < 100 %	68.17	pass
SD of peptide depletion of the PC replicates	<14.9 %	0.05	pass
SD of peptide depletion of the TI replicates	<14.9 %	0.54	pass

 

Table 9: 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	0.5003	pass
mean peptide concentration of RC C (PC)	0.45 ≤ x ≤ 0.55 mM	0.4987	pass
mean peptide concentration of RC C (TI)	0.45 ≤ x ≤ 0.55 mM	0.5045	pass
CV of the peak area of RC B	<15 %	0.48	pass
CV of the peak area of RC C (PC)	<15 %	0.47	pass
CV of the peak area of RC C (TI)	<15 %	0.32	pass
mean peptide depletion of the PC	40.2 % < x < 69.0 %	56.62	pass
SD of peptide depletion of the PC replicates	<11.6 %	1.13	pass
SD of peptide depletion of the TI replicates	<11.6 %	0.15	pass

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.

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, according to OECD TG 442C, the test item was dissolved in N.N-dimethylformamide (DMF) based on the results of the pre-experiments. Based on a molecular weight of 324.82 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 solution of the test item 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. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item 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. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis.

A minor co-elution of the test item with the lysine peptide peak was observed. The peak area determined in the co-elution controls corresponds to 0.0104 mM of lysine peptide. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of the cysteine peptide was ≤ 13.89 % (5.58 %). According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

Since precipitation was observed for the lysine and cysteine peptide samples containing also test item after the incubation period, no final prediction can be made.

In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made.