Ammonium undecafluorohexanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Mar - 03 Apr 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:

04 Feb 2015

Deviations:

yes

Remarks:

No reference to historical positive control and reference control data.

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium undecafluorohexanoate

EC Number:

244-479-6

EC Name:

Ammonium undecafluorohexanoate

Cas Number:

21615-47-4

Molecular formula:

C6H4F11NO2

IUPAC Name:

2,2,3,3,4,4,5,5,6,6,6-Undecafluorohexanoic acid, ammonium salt (1:1)

Test material form:

solid

In chemico test system

Details on the study design:

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular reaction event of the skin sensitisation adverse outcome pathway, 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 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model. This allows the assigning of the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: Scrum
Synthetic cysteine-containing peptide:
- Lot. number: 15611
- Purity: 92.81%
Synthetic lysine-containing peptide:
- Lot. number: 16335
- Purity: 93.21%

SOLVENT CONTROL AND ASSESSMENT OF TEST ITEM SOLUBILITY
- Solvent: acetonitrile
- Lot. number: KPE0556
- Purity: 100%
The test substance was dissolved at the concentration of 100 mM in acetonitrile which is recommended as a solvent in OECD TG442C. The lot of the acetonitrile used in this study was confirmed not to affect the stability of the peptides.

POSITIVE CONTROL
- Substance: cinnamaldehyde
- Lot. number: ECL6837
- Purity: 99.1%
The positive control was prepared at a concentration of 100 mM.

REFERENCE CONTROL PREPARATION
For each peptide reference controls were prepared by mixing 750 μL of the 0.667 mM standard stock solution with 250 μL of acetonitrile.

PEPTIDE STOCK SOLUTION PREPARATION
Synthetic 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 OF THE TEST SUBSTANCE WITH THE PEPTIDE SOLUTIONS
- Peptide to test substance ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance/positive control); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance/positive control)
Test substance reaction solutions were visually inspected immediately after the preparation and 22 hours after the preparation. No suspension or precipitation were observed.
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 24 h prior to initiation of the analysis run

NUMBER OF REPLICATES
for each peptide: triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Hitachi High-Technologies L-2130 pump A and B, L-2200 auto sampler, L-2400 wavelength detector, L-2300 column oven and EZChrom Elite data processor
- Column: L-column2 ODS (2.1mm I.D. x 100 mm, CERI)
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in aqueous solution
B: 0.085% (v/v) trifluoracetic acid in acetonitrile solution
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10,10.5, 12.5, 13, 20
%A: 90, 75, 10, 10, 90, 90
% B: 10, 25, 90, 90, 10, 10
- Detector Wavelength: 220 nm
- Calibration standard concentrations of both peptides: 0.267, 0.134, 0.0667, 0.0334 and 0.0167 mM
- Column temperature: 30 °C

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: ≥ 24 h incubation

Parameter:

other: % depletion of cysteine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

1.3 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: ≥ 24 h incubation

Parameter:

other: % depletion of lysine-containing peptide

Remarks:

(mean value of 3 replicates)

Value:

100 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: ≥ 24 h incubation

Parameter:

other: % overall mean depletion

Value:

50.7 %

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Other effects / acceptance of results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 3. Mean peptide depletion of cysteine-containing peptide.

	Peak area				Peptide depletion (%)
	Individual	Mean	SD	CV (%)	Individual	Mean	SD	CV (%)
Positive control	534167	530888	6728	1.3	74.3	74.5	0.3	0.4
	535349				74.3
	523149				74.8
Test substance	2067534	2053358	15204	0.7	0.6	1.3	0.7	53.8
	2037301				2.0
	2055238				1.2

 CV: Coefficient of Variation

 

Table 4. Mean peptide depletion of lysine-containing peptide.

	Peak area				Peptide depletion (%)
	Individual	Mean	SD	CV (%)	Individual	Mean	SD	CV (%)
Positive control	806013	834673	30312	3.6	54.4	52.8	1.7	3.2
	866403				51.0
	831603				52.9
Test substance	0	0	0	-	100.0	100.0	0.0	0.0
	0				100.0
	0				100.0

 CV: Coefficient of Variation

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitising potential based on the key event “protein reactivity”

Conclusions:

Under the conditions of the Direct Peptide Reactivity Assay the test substance showed a mean cystein and lysine peptide depletion of 50.7%, thus the peptide reactivity is classified as high and the prediction for skin sensitivity is positive. 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 showed reactivity towards selected proteins. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.