Ammonium undecafluorohexanoate

Endpoint summary

Description of key information

Skin sensitisation (OECD 442C and 442E): positive

WoE conclusion from in vitro skin sensitisation battery: skin sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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)

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

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

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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

The skin sensitising potential of the test substance was evaluated in a combination of non-animal methods (in chemico and in vitro). Two key events of skin sensitisation a) molecular interaction with skin proteins and b) activation of dendritic cells were addressed.

a) In chemico: Molecular interaction with skin proteins

The protein reactivity of the test substance was investigated in a Direct Peptide Reactivity Assay (DPRA) (Tsubokura, 2017) performed according to OECD guideline 442C and in compliance with GLP. The test substance was dissolved in acetonitrile to prepare a 100 mM solution. The solubility in acetonitrile was confirmed. Stock solutions of cysteine- and lysine-containing synthetic peptides with a purity of about 93% were freshly prepared just before incubation with the test substance. The cysteine and lysine peptide solutions were incubated in HPLC vials with the test substance or the positive control cinnamic aldehyde at 1:10 (5 mM cysteine and 5 mM test substance/positive control) and 1:50 ratios (0.5 mM lysine and 25 mM test substance/positive control), respectively. After an incubation period of a minimum of 24 h at 25 °C in an HPLC autosampler, HPLC-UV analysis for the cysteine and lysine peptides were performed. All analytical acceptance criteria for each peptide run were met and the positive control proved the validity of the test. In presence of the test substance the mean depletion of cysteine and lysine peptide was 1.3% and 100.0%, respectively. The overall mean percent cysteine and lysine depletion was 50.7%, and therefore the test substance is allocated to the “high reactivity” class (mean % depletion > 42.47%) and considered to be positive for molecular interaction with skin proteins.

 

 b) In vitro: Activation of dendritic cells

The dendritic cell response of a 50% solution of the test substance was investigated in a human Cell Line Activation Test (h-CLAT) according to OECD guideline 442E and in compliance with GLP ( Maeda, 2017). The doses for the main experiment were previously determined by a viability test with propidium iodide. Cell viability was 85.5 and 3.1% at test substance concentrations of 1000 and 2500 mg/mL, respectively, and the CV75 was calculated to be 1123.6 μg/mL. Therefore, 1348.3 μg/mL (1.2 X CV75) was selected as the highest dose. In the main experiment human THP-1 cells were exposed to 376.3, 451.5, 541.8, 650.2, 780.2, 936.3, 1123.6 and 1348.3 µg/mL test substance in DMSO for 24 h. Following the exposure, the cells were stained with FITC-labeled anti-CD86, CD54 antibody or mouse IgG1 (isotype control) and propidium iodide. Relative fluorescence intensity of the surface markers and viability of cells were measured in a flow cytometer. In two independent runs, the maximum RFI of CD54 was over 200 (maximum values of 232.9 and 231.5) while the maximum RFI of CD86 was less than 150 (maximum values of 64.2 and 97.4). All acceptance criteria were met. Therefore, the h-CLAT prediction for activation of dendritic cells is considered to be positive. The study was conducted with a 50% solution of the test substance. Although the results are considered valid as they were positive even at lower concentrations.

 

Overall conclusion

Positive results were observed in both of the in vitro test battery assays performed, therefore the test substance is considered to be a skin sensitiser.

Justification for classification or non-classification

The available data on skin sensitisation of the test substance meet the criteria for classification as Skin Sens. 1 (H317) according to Regulation (EC) 1272/2008.