N,N-bis(2-ethylhexyl)formamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 - 08 Apr 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

adopted in 2020

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

N,N-bis(2-ethylhexyl)formamide

EC Number:

860-695-9

Cas Number:

88247-41-0

Molecular formula:

C17H35NO

IUPAC Name:

N,N-bis(2-ethylhexyl)formamide

Test material form:

liquid

In chemico test system

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

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 UV detection at 220 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.

TEST SYSTEM:
- Supplier: JPT Peptide Technologies GmbH, Berlin, Germany
- Cysteine-containing peptide:
Ac-RFAACAA-COOH
Batch number: 111016HS_MHeW1220
Molecular weight: 750.9 g/mol
Purity: >98%
- Lysine-containing peptide:
Ac-RFAAKAA-COOH
Batch number: 020517HS_MHeW1220
Molecular weight: 775.9 g/mol
Purity: 98.04%


CONTROL:
- A co-elution control, test item at a concentration of 100 mM in acetonitrile and the respective buffer solution (phosphate buffer for Cys peptide, ammonium acetate buffer for Lys peptide) without synthetic peptide, was prepared.
- Reference controls were prepared by mixing the respective synthetic peptide with acetonitrile
- Calibration solutions were prepared in order to generate a calibration curve.

POSITIVE CONTROL FOR CYSTEINE-CONTAINING AND FOR LYS-CONTAINING PEPTIDE:
- Substance: Cinnamic aldehyde
- Batch number: MKCB9907
- Purity: 99.1%
- Expiry date: 30 Nov 2021


POSITIVE CONTROL PREPARATION:
The positive control was prepared at a concentration of 100 mM in acetonitrile. 250 µL of the solution were mixed with 750 µL of the respective peptide solution (0.667 mM SPCC or SPCL).

TEST SUBSTANCE PREPARATION:
The test substance was prepared as a 100 mM formulation in acetonitrile (ACN) freshly for each reactivity assay.
For both the cysteine and lysine reactivity assays 41.88 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1554 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the clear solution being formed was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.

PEPTIDE STOCK SOLUTION PREPARATION:
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM; 10.2 mg synthetic peptide containing cysteine was dissolved in 20.36 mL phosphate buffer pH 7.5.
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM; 10.4 mg was dissolved in 20.08 mL ammonium acetate buffer pH 10.2.

INCUBATION CONDITIONS:
- Peptide ratios: Cysteine-containing peptide: 1:10; lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: 23.9 - 30 h

PRECIPITATION:
Prior to HPLC analysis the samples were visually inspected. If precipitation or phase separation was observed, the samples were centrifuged. In the current assay, neither precipitation nor phase separation were observed.

NUMBER OF REPLICATES:
Samples for treatment and control were prepared in triplicate for each peptide.

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector (Waters, Milford, Massachusetts, USA)
- Column: Zorbax SB C18, 3.5 μm, 100 x 2.1 mm (Agilent Technologies, Santa Clara, California, USA) with a guard column (SecurityGuard™ cartridge for C18, 4 × 2.0 mm, Phenomenex, Torrance, California, 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 min
% B: 10, 25, 90, 90, 10, 10 for cysteine and 10, 20, 90, 90, 10, 10 for lysine
- Injection volume: 5 μL
- Column temperature: 30 °C

STANDARD CALIBRATION CURVES:
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.9999. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.
The correlation coefficient (r2) of the SPCL standard calibration curve was 0.9995. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.


ACCEPTABILITY CRITERIA:
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have a r2 > 0.99.
b) The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8% and 100% for SPCC and between 40.2% and 69.0% for SPCL.
c) The maximum standard deviation (SD) for the positive control replicates had to be <14.9% for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide peak areas for the nine Reference Controls B and C in ACN had to be <15.0%.
 
The following criteria had to be met for a test item’s results to be considered valid:
a) The maximum SD for the test item replicates had to be <14.9% for the Percent Cysteine Depletion and <11.6% for the Percent Lysine Depletion.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.


DATA EVALUATION:
- Cys and Lys peptide detection wavelength: 220 and 258 nm
The concentration of synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:
Percent Peptide Depletion= [1−(Peptide Peak Area in Replicate Injection (at 220 nm) / Mean Peptide Peak Area in Reference Controls (at 220 nm))]×100
In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90% < mean area ratio of control samples < 110% gives a good indication that co-elution has not occurred.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

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 peptide depletion of the positive control for the cysteine peptide was 69.9 %. The mean peptide depletion of the positive control for the lysine peptide was 54.2%.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

1.4 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

phase separation observed

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0.8 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

phase separation observed

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

TEST-SPECIFIC CONFOUNDING FACTORS
After the incubation period, phase separation was observed in the co-elution control samples and in the test item samples for both peptides. It cannot be sure how much test item remained in the solution to react with the respective peptide. Consequently, this negative result is uncertain and should be interpreted with due care.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes, the mean percent peptide depletion value were between 60.8 and 100% for the cysteine containing peptide (69.9%) and between 40.2 and 69.0% for the lysine containing peptide (54.2%).
- Acceptance criteria met for reference controls A to C: Yes, the mean peptide concentration of the reference control A was 0.511 ± 0.002 mM for the cysteine containing peptide and 0.511 ± 0.004 for the lysine containing peptide and fell both into the accepted range of 0.50 ± 0.05.
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes, the mean peptide concentration of the co-elution control was 0.501 ± 0.005 mM for the cysteine containing peptide and 0.505 ± 0.007 for the lysine containing peptide and fell both into the accepted range of 0.50 ± 0.05.
- Acceptance criteria met for variability between replicate measurements: Yes, the maximum standard deviation for the positive control replicate were < 14.9% for percent cysteine peptide depletion (0.4%) and < 11.6% for percent lysine peptide depletion (0.9%). The coefficient of variation of peptide areas for the reference controls B and C in acetonitrile were < 15.0% (0.8% for the cysteine containing peptide and 2.0 % for the lysine containing peptide).

Any other information on results incl. tables

Table 1: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Substance	SPCC depletion (%)					SPCL depletion (%)					Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Sample 1	Sample 2	Sample 3	Mean	± SD	Sample 1	Sample 2	Sample 3	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test item	0.9	0.8	2.3	1.40%	± 0.8%	1.1	0.0	1.4	0.80%	± 0.7%	1.10%	Negative: No or minimal reactivity
Positive control Cinnamic aldehyde	69.7	69.7	70.4	69.90%	± 0.4%	54.8	54.7	53.1	54.20%	± 0.9%	62.10%	Positive: high reactivity

SD = Standard Deviation

Table 2: Acceptability of the Direct Peptide Reactivity Assay (DPRA)

	Cysteine reactivity assay		Lysine reactivity assay
	Acceptability criteria	Results for SPCC	Acceptability criteria	Results for SPCL
Correlation coefficient (r^2) standard calibration curve	>0.99	0.9999	>0.99	0.9995
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.511 ± 0.002	0.50 ± 0.05	0.511 ± 0.004
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.501 ± 0.005	0.50 ± 0.05	0.505 ± 0.007
CV (%) for RC samples B and C	<15.0	0.8	<15.0	2.0
Mean peptide depletion cinnamic aldehyde (%)	60.8 - 100	69.9	40.2 - 69.0	54.2
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.4	<11.6	0.9
SD of peptide depletion for the test item (%)	<14.9	0.8	<11.6	0.7

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:

other: inconclusive based on the key event “protein reactivity”

Conclusions:

There is regulatory acceptance in the EU for the application of the Direct peptide reactivity assay to address key event 1, peptide/protein binding, in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance did not show reactivity towards selected proteins. However, phase separation was observed after incubation with the test item and both peptides. It cannot be sure how much test item remained in the solution to react with the peptide. Consequently, the test result for the endpoint peptide/protein binding is inconclusive.
Further evaluation and/or data generation is required to decide on the non-classification or classification as skin sensitiser of the test substance.