2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

adopted in June 2019

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.59

Version / remarks:

adopted in February 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

1

Reference substance name:

2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

EC Number:

206-406-6

EC Name:

2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

Cas Number:

335-99-9

Molecular formula:

C7H4F12O

IUPAC Name:

2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptan-1-ol

Test material form:

liquid

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.
For comparison, tests were performed with the test item, the vehicle (solvent control = negative control) and the known sensitizer Cinnamic aldehyde (positive control).

The DPRA quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 +/-2.5 ºC. Relative peptide concentration is measured by reversed phase (C18) high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 and 258 nm. The synthetic peptides contain phenylalanine to aid in the detection.
The test item was dissolved and tested according to the given test procedure. Cinnamic aldehyde was used as positive control at a concentration of 100 mmol/L in acetonitrile.

Cysteine and lysine peptide solutions were incubated at 1:10 and 1:50 ratio in glass auto sampler vials with the test item solution for 24 hours at 25 ± 2.5 °C in the dark. Samples were visually inspected for precipitation or phase separation before HPLC analysis. The test item was analyzed in triplicate for both peptides. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours. HPLC analysis for the cysteine and lysine peptides were performed on separate days with the test item solutions freshly prepared for both assays on each day.

The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards. Percent peptide depletion is calculated according to OECD Guideline.

Acceptance criteria:
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.
The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should 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 should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

According to the study protocol the mean percent cysteine and percent lysine depletion value was calculated for the test item and the positive control. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion can be used to support the discrimination between skin sensitizers and non-sensitizers in the framework of an IATA.

Results and discussion

Positive control results:

The positive control cinnamic aldehyde led to a depletion of 69.52 % cysteine peptide and 55.87 % lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.4% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates was < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: mean of 2 runs

Parameter:

other: % depletion in the cysteine 1:10/lysine 1:50 prediction model:

Value:

1.43 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

DPRA prediction: negative

Results 2

Parameter:

cysteine depletion

Value:

2.86 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Parameter:

lysine depletion

Value:

0 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The acceptance criteria for a DPRA test to be considered valid were met.
The linearity of the standard calibration curve was r2 = 1.0000 for cysteine peptide and r2 = 0.9999 for lysine peptide. Hence the requirement of r2 > 0.99 was met.
The mean peptide concentrations of reference control A were 0.505 mM for cysteine peptide or 0.493 mM for lysine peptide, and, hence well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C was <15.0%.
The acceptance criteria of validity described were fulfilled in this test system.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes

The test item was completely dissolved and stable in acetonitrile at a concentration of 100 mM. The formulation analysis performed at LPT resulted in actual levels of 1H,1H,7H-perfluoroheptane-1-ol of 93.9% of the nominal concentrations determined via GC-FID.
No precipitate in the reaction mixture at the end of the incubation time and no co-elution were observed.

Any other information on results incl. tables

Results of the DPRA:

Test item	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	reactivity class	DPRA prediction
positive control (cinnamic aldehyde)	69.52	55.87	62.70	high	positive
test item	2.86	0	1.43	minimal	negative

No precipitation was observed after the incubation period.

Applicant's summary and conclusion

Interpretation of results:

other: negative

Executive summary:

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

Since the test item visually appeared a clear solution in acetonitrile at the test concentration of 100 mmol/L this solvent was used in the DPRA. No precipitaton of the test item occured after incubation and no co-elution was observed. The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. No relevant depletion of cysteine and lysine peptides became obvious in the DPRA (1.43 % peptide depletion). According to the prediction model “minimal reactivity” was derived for the test item in water, leading to a DPRA prediction of “negative“.