6-hexyltetrahydro-2H-pyran-2-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

17 April 2018 - 01 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of a weight of evidence approach.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

4 February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

A validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as one of the non-animal tests to be performed as part of a weight of evidence approach.

Test material

Test material information

Constituent 1

Reference substance name:

Tetrahydro-6-propyl-2H-pyran-2-one

EC Number:

211-820-5

EC Name:

Tetrahydro-6-propyl-2H-pyran-2-one

Cas Number:

698-76-0

Molecular formula:

C8H14O2

IUPAC Name:

6-propyloxan-2-one

Test material form:

liquid

Details on test material:

Appearance: Clear colourless to pale yellow liquid
Storage: In refrigerator (2-8°C)

In chemico test system

Details on the study design:

TEST ITEM PREPARATION
Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvent was evaluated: acetonitrile (ACN).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 23.54 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1655 μL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution 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.

TEST SYSTEM
Synthetic peptides containing cysteine (SPCC) (Ac-RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac-RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL. Rationale: Recommended test system in the international OECD guideline for DPRA studies.
Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test substance samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 24.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
Prior to HPLC-PDA analysis the samples were visually inspected for precipitation.

POSITIVE CONTROL
Cinnamic aldehyde
- Purity: 98.4%
- Batch: MKBP1014V
- Expiry of batch: 31 May 2018

DATA EVALUATION:
The concentration of SPCC or 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))]x100

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%
DATA INTERPRETATION
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test substance. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see Table 1), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an 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 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.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: Mean SPCC depletion(%)

Value:

1.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 0.8%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 74.0% ± 0.2%.

Remarks on result:

other: SD: 0.3%

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: Mean percentage SPCC:

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 1.3%

Positive controls validity:

valid

Remarks:

Mean percentage SPCL: 53.2% ± 1.8%.

Remarks on result:

other: SD: 0.7%

Other effects / acceptance of results:

All acceptability criteria were met and therefore the DPRA was considered to be valid.
See Table 2 & 3 "any other information on results incl. tables" for acceptibility criteria.

Any other information on results incl. tables

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	0.996	>0.99	0.997
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.508 ± 0.006	0.50 ± 0.05	0.498 ± 0.007
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.505 ± 0.005	0.50 ± 0.05	0.498 ± 0.004
CV (%) for RC samples B and C	<15.0	0.8	<15.0	1.3
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	74.0	40.2-69.0	53.2
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.2	<11.6	1.8
SD of peptide depletion for the test item (%)	<14.9	0.3	<11.6	0.7

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

Table 3: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Item

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Tetrahydro6propyl-2Hpyran-2-one (delta octalactone)	1.2%	±0.3%	2.5%	±0.7%	1.9%	Negative: No or minimal reactivity

SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:

other: Study cannot be used for classification independently, but in a WoE for the end point Skin Sensitisation

Conclusions:

Delta octalactone was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.