Dec-9-en-1-ol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2017 - 01 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The DPRA assay 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 weight of evidence.

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)

Test material

Test material information

Constituent 1

Reference substance name:

Dec-9-en-1-ol

EC Number:

235-878-6

EC Name:

Dec-9-en-1-ol

Cas Number:

13019-22-2

Molecular formula:

C10H20O

IUPAC Name:

dec-9-en-1-ol

Test material form:

liquid

Details on test material:

- Physical appearance: clear colourless liquid
- Storage conditions: at room temperature, protected from light

Specific details on test material used for the study:

At a concentration of 100 mM, the test item was not soluble in MQ, MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and DMSO/ACN (1:9, v/v), but was soluble in ACN. Therefore this solvent was used to dissolve the test item in this DPRA study.

In chemico test system

Details on the study design:

TEST ITEM PREPARATION
No correction for the purity/composition of the test item was performed.
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 neither cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), MQ/ACN (1:1, v/v), isopropanol, acetone, acetone/ACN (1:1, v/v) and dimethylsulfoxide (DMSO)/ACN (1:9, v/v).
Test item stock solutions were prepared freshly for each reactivity assay.
For both the cysteine and lysine reactivity assay 22.50 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1440 μL ACN 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 of SPCC is 750.9 g/mol, and 775.9 g/mol for SPCL. The peptides were stored in the freezer (<-15°C) for a maximum of 6 months.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Calibration curve SPCC and SPCL: according to guideline
- Incubation: After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item 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.
- Analysis: All samples were analyzed according to the HPLC-PDA method.

POSITIVE CONTROL: Cinnamic aldehyde
- Purity: 98.4%

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))]*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.

DATA INTERPRETATION (see also 'Other information on materials and method')
The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. 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 was used to support the discrimination between a skin sensitizer and a non-sensitizer.

ACCEPTABILITY CRITERIA:
The acceptability criteria of the test are summarized in table 2 ('other information on results').

Results and discussion

Positive control results:

The positive control had a mean SPCC depletion of 77.3 ± 0.1% and a mean SPCL depletion of 53.4 ± 1.2%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC mean depletion (%)

Value:

7.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD: 0.7%

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL mean depletion (%)

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SD: 1.4%

Other effects / acceptance of results:

- In both the Cysteine Assay as the Lysine Assay, no precipitation of the test item was observed.
- For details on results see table 2 and 3 in 'any other information on results'.

In both the cysteine reactivity assay and the lysine reactivity assay, all acceptability criteria were met and the assays are considered valid (see table 4).

Any other information on results incl. tables

Table 2 Acceptability of the DPRA assay

	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	0.9990	>0.99	0.998
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.503 ± 0.005	0.50 ± 0.05	0.482 ± 0.001
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.504 ± 0.000	0.50 ± 0.05	0.486 ± 0.008
CV (%) for RC samples B and C	<15.0	0.8	<15.0	1.2
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	77.3	40.2-69.0	53.4
SD of peptide depletion cinnamic aldehyde (%)	<14.9	0.1	<11.6	1.2
SD of peptide depletion for Florantone T (%)	<14.9	0.7	<11.6	1.4

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation; NA = Not Applicable.

* For calculation of the RC-C mean and the CV, the RCcysC-3 value was excluded (outlier due to injection error). As a result, no SD could be calculated for the mean of the RCcysC.

Table 3 SPCC and SPCL depletion and reactivity classification for Florantone T

Test item	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	Reactivity class
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Trepanol	7.3%	±0.7%	1.6%	±1.4%	4.4%	Negative: No or minimal reactivity

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Study is part of a weight of evidence approach and is not used for classification on its own.

Conclusions:

Trepanol was negative in a DPRA performed according to OECD guideline 442C and GLP principles. The test item was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. The study cannot be used stand-alone to draw a conclusion on classification for skin sensitisation.