Dec-9-en-1-ol

Endpoint summary

Description of key information

According to the OECD TG studies 442C and 442D carried out under GLP conditions, the test substance is negative for sensitising effects.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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.

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)

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.

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').

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%.

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%

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).

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

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 April 2018 - 18 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd. 22 January 2018

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The purpose of this study was to evaluate the ability of Trepanol to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. Activation of this pathway can lead to skin sensitisation.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Number of replicates: two independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

CONTROLS:
Positive control: ethylene dimethacrylate glycol (purity: 98.3%), 7.8-250 μM, tested in triplicate, DMSO was used as a vehicle;
Negative control: DMSO (1% in exposure medium);
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values.

TEST SYSTEM:
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (the KeratinoSens™ cell line). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25). One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+12 in experiment 1 and P+17 in experiment 2.

TEST ITEM PREPARATION:
The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (clear colorless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.

TEST CONCENTRATIONS:
- Both experiments: 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM

MEDIA:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

TREATMENT OF CELLS:
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. In total 2 valid experiments were performed.

LUCIFERASE ACTIVITY MEASUREMENT:
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time two seconds).

CYTOTOXICITY ASSESSMENT:
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

The EC1.5 of the positive control was between 5 and 125 μM (38 μM and 53 μM in experiment 1 and 2, respectively). A dose related response was observed and the induction at 250 μM was higher than 2-fold (3.24-fold and 2.80-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.37

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1.33

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

- In the first experiment, the test item showed toxicity (IC30 value of 337 μM, IC50 value of 427 μM). No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.37 and therefore no EC1.5 could be calculated.
- In the second experiment, the test item showed toxicity (IC30 value of 301 μM, IC50 value of 358 μM). No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. The Imax was 1.33 and therefore no EC1.5 could be calculated.

- In both experiments, no precipitation was observed at the start and end of the incubation period in the 96-well plates.

Both experiments passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 μM (38 μM and 53 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.24-fold and 2.80-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (10% and 11% in experiment 1 and 2, respectively).

Table 1 Overview Luminescence Induction and Cell Viability of the test item in Experiment 1 and 2

Concentration (µM)	0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
Exp 1 luminescence	1.21	1.20	1.10	1.23	1.24	1.30	1.37	1.30	1.31	0.25	0.00	0.21
Exp 1 viability (%)	98.8	93.0	96.5	97.6	89.7	87.4	94.1	85.8	89.2	33.8	-1.1	26.8
Exp 2 luminescence	1.00	1.01	1.02	1.16	1.18	1.27	1.25	1.33	1.26	0.03	0.00	0.00
Exp 2 viability (%)	103.2	100.1	97.9	95.1	96.8	93.9	92.3	93.6	87.9	0.0	0.1	0.4

Table 2 Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.12	1.20	1.40	1.84^***	2.05^***	3.24^***
Exp 1 viability (%)	89.6	88.9	90.6	94.2	94.4	99.7
Exp 2 luminescence	1.05	1.16	1.32	1.58^***	2.04^***	2.80^***
Exp 2 viability (%)	93.4	98.2	101.3	102.8	108.1	107.6

^***p<0.001 Student’s t test

Table 3 Overview EC_1.5, I_max, IC₃₀ and IC₅₀ Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.37	337	427
Test item Experiment 2	NA	1.33	301	358
Pos Control Experiment 1	38	3.24	NA	NA
Pos Control Experiment 2	53	2.80	NA	NA

NA = Not applicable

Interpretation of results:

study cannot be used for classification

Remarks:

This study is part of a weight of evidence approach on which the classification is based.

Conclusions:

Based on the outcome of a KeratinoSens™ assay performed according to OECD guideline and GLP principles, Trepanol is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)