4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 - 09 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate of the Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

EC Number:

235-985-8

EC Name:

4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

Cas Number:

13080-86-9

Molecular formula:

C27H26N2O2

IUPAC Name:

4,4'-[isopropylidenebis(4,1-phenyleneoxy)]dianiline

In chemico test system

Details on the study design:

TEST SYSTEM
- Supplier: JPT Peptide Technologies GmbH, Berlin, Germany

- Cysteine-containing peptide:
Amino acido sequence: Ac-RFAACAA-COOH
Batch number: 111016HS_MHeW0217
Purity: > 98%

- Lysine-containing peptide:
Amino acido sequence: Ac-RFAAKAA-COOH
Batch number: 220114HSDW_W0217
Purity: > 98%

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

REFERENCE CONTROL (RC)
Concentration: 0.5 mM
- RC A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification.
- RC B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time.
- RC C (vehicle control) 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, RC C was used to calculate PPD.

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.

POSITIVE CONTROL
- Substance: cinnamic aldehyde (in acetonitrile)
- Supplier: Sigma Aldrich, Steinheim, Germany
- Batch number: MKBP1014V
- Purity: > 98.4%

POSITIVE CONTROL PREPARATION
The positive control was prepared at a stock concentration of 100 mM (in acetonitrile).

TEST SUBSTANCE PREPARATION
The test substance was prepared at a stock concentration of 100 mM (in acetonitrile).

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM

Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS
- Peptide solutions:
Cysteine-containing peptide: 750 µL peptide and 50 µL test item/ positive control solution and 200 µL acetonitrile
Lysine-containing peptide: 750 µL peptide and 250 µL test item/ positive control
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: 22 - 24 h

NUMBER OF REPLICATES
triplicates

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 µm (Agilent Technologies, Santa Clara, USA)
- Guard column: SecurityGuardTM cartridge for C18, 4.0 x 2.0 mm (Phenomenex, Torrance, USA)
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in Milli-Q water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20 (end run)
% A: 90, 75, 10, 10, 90
% B: 10, 25, 90, 90, 10
- Wavelength for detection: 220 and 258 nm
- Injection volume: 4 μL
- Column temperature: 30 °C

Results and discussion

Positive control results:

The positive control induced a positive response in both peptide runs, as the mean peptide depletions were 57.35% and 70.37% for lysine and cysteine, respectively.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: test substance

Parameter:

other: % depletion of cysteine-containing peptide (mean value of 3 replicates)

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(reference controls)

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: positive control

Parameter:

other: % depletion of cysteine-containing peptide (mean value of 3 replicates)

Value:

73.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(reference controls)

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: test substance

Parameter:

other: % depletion of lysine-containing peptide (mean value of 3 replicates)

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(reference controls)

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 4

Run / experiment:

other: positive control

Parameter:

other: % depletion of lysine-containing peptide (mean value of 3 replicates)

Value:

58.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(reference controls)

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
Precipitation was observed upon addition of the test chemical to the SPCC (Synthetic Peptide Containing Cysteine) and SPCL (Synthetic Peptide Containing Lysine) peptide solutions and in the the co-elution controls.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, in separate experiment (do data available)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
mean peptide concentration of reference control A and C (cysteine peptide run) = 0.503 ± 0.005 and 0.501 ± 0.010, respectively (acceptable: 0.50 ± 0.05 mM)
mean peptide concentration of reference control A and C (lysine peptide run) = 0.548 ± 0.003 and 0.546 ± 0.021, respectively (acceptable: 0.50 ± 0.05 mM)

- Acceptance criteria met for positive control (PC): yes
mean peptide depletion of the PC (cysteine peptide run) = 73.6% (range: 60.8% - 100%)
mean peptide depletion of the PC (lysine peptide run) = 58.4% (range: 40.2% - 69.0%)

- Acceptance criteria met for variability between replicate measurements: yes
SD of peptide depletion of the PC replicates (cysteine peptide run) = 1.6 (< 14.9%)
SD of peptide depletion of the test substance replicates (cysteine peptide run) = 1.1 (< 14.9%)
SD of peptide depletion of the PC replicates (lysine peptide run) = 0.4 (< 11.6%)
SD of peptide depletion of the test substance replicates (lysine peptide run) = 1.3 (< 11.6%)

Any other information on results incl. tables

Table 2: Mean peptide depletion of cysteine-containing peptide

Cysteine peptide
Sample	Peak area at 220 nm [µAU]	Peptide concentration [mM]	Peptide depletion [%]	Mean peptide depletion [%]	SD of peptide depletion [%]
Positive control	1002742	0.163	72.9	73.6	1.6
	1021122	0.138	72.4
	921906	0.124	75.3
Test substance	3505855	0.505	0.0	1.1	1.1
	3437717	0.495	1.2
	3402865	0.49	2.2

Table 3: Mean peptide depletion of lysine-containing peptide

Lysine peptide
Sample	Peak area at 220 nm [µAU]	Peptide concentration [mM]	Peptide depletion [%]	Mean peptide depletion [%]	SD of peptide depletion [%]
Positive control	1006860	0.225	54.8	58.4	0.4
	1020611	0.228	58.2
	1024158	0.229	58.1
Test substance	2300770	0.532	2.5	1.1	1.3
	2343450	0.542	0.7
	2367102	0.548	0.0

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitising potential based on the key event “protein reactivity”

Conclusions:

Under the conditions of the test, the test substance did not show reactivity towards selected peptides. However, since precipitation was observed upon addition of the test chemical to the Synthetic Peptide Containing Cysteine and Synthetic Peptide Containing Lysine peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.
In general, the result of this test alone does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.