Cholest-5-en-3-β-yl acetate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 January 2018 - 23 January 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 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)

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 weight of evidence.

Test material

Test material information

Constituent 1

Reference substance name:

Cholest-5-en-3-β-yl acetate

EC Number:

210-066-4

EC Name:

Cholest-5-en-3-β-yl acetate

Cas Number:

604-35-3

Molecular formula:

C29H48O2

IUPAC Name:

(1R,3aS,3bS,7S,9aR,9bS,11aR)-9a,11a-dimethyl-1-[(2R)-6-methylheptan-2-yl]-1H,2H,3H,3aH,3bH,4H,6H,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-7-yl acetate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White crystalline powder
- Storage condition: At room temperature

In chemico test system

Details on the study design:

TEST ITEM PREPERATION
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 solvents were evaluated: acetonitrile (ACN), Milli-Q water (MQ), ACN/MQ (1:1, v/v), isopropanol, acetone/ACN (1:1, v/v) dimethylsulfoxide (DMSO)/ACN (1:9, v/v) and ethanol.
Test item stock solutions were prepared freshly for each reactivity assay. For both the cysteine and lysine reactivity assay 7.65 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1784 µL EtOH after vortex mixing and 1 minute of sonication to obtain a 10 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.

#At a concentration of 100 mM, Cholesteryl acetate was not soluble in any of the solvents tested. At a concentration of 10 mM, the test item was soluble in ethanol (EtOH). Therefore, the Direct Peptide Reactivity Assay (DPRA) study was performed using a 10 mM test item solution prepared in this solvent.

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, 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 25 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. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature.
- Analysis: All samples were analyzed according to the HPLC-PDA method presented in Table 1 ('Other information on methods and materials').
The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 ('Other information on materials and methods').

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))]*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 Table 3 '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.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Cysteine Reactivity Assay

Parameter:

other: SPCC mean percentage

Value:

15.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 0.9%

Positive controls validity:

valid

Remarks:

Mean percentage SPCC: 76.2% ± 1.2%.

Remarks on result:

other: SD: 3.4%

Results 2

Key result

Run / experiment:

other: Lysine Reactivity Assay

Parameter:

other: SPCL mean percentage

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

CV between reference controls: 1.6%

Positive controls validity:

valid

Remarks:

mean percentage SPCL: 53.6% ± 2.3%

Remarks on result:

other: SD: 0%

Other effects / acceptance of results:

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.
See table 4 and 5 in " any other information on results incl. table" for results and acceptability of DPRA

Any other information on results incl. tables

Table 4: 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	0.997	>0.99	0.9991
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.502 ± 0.005	0.50 ± 0.05	0.471 ± 0.013
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.494 ± 0.002	0.50 ± 0.05	0.469 ± 0.003
Mean peptide concentration RC-CEtOHsamples (mM)	0.50 ± 0.05	0.499 ± 0.003	0.50 ± 0.05	0.458 ± 0.011
CV (%) for RC samples B and C	<15.0	0.9	<15.0	1.6
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	76.2	40.2-69.0	53.6
SD of peptide depletion cinnamic aldehyde (%)	<14.9	1.2	<11.6	2.3
SD of peptide depletion for the test item (%)	<14.9	3.4	<11.6	0.0

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

Table 5: 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
Cholesteryl acetate	15.9%	±3.4%	0.0%	±0.0%	7.9%	Positive: Low reactivity

SD = Standard Deviation.

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:

Cholesteryl acetate was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In an in chemico study, performed according to OECD guideline 442C and GLP principles, the reactivity of Cholesteryl acetate towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined to assign the test chemical to one of four reactivity classes used to support the discrimination between skin sensitisers and non skin sensitisers. Following incubation of the test substance with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in the prediction model.

Ethanol was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. Cinnamic aldehyde was used as a positive control. The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test substance, were within the acceptability criteria for the DPRA assay. Therefore, the study was considered to be valid.

In the cysteine reactivity assay the test item showed 15.9% SPCC depletion while in the lysine reactivity assay the test item showed 0.0% SPCL depletion. The mean of the SPCC and SPCL depletion was 7.9% and as a result the test item was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Therefore, the test item was considered to be positive in the DPRA. However, since precipitation was observed upon addition of the test item to the SPCC and SPCL peptide solutions, one cannot be sure how much test item remained in the solution to react with the peptides. In addition, the DPRA study was performed using a test item concentration of 10 mM. Consequently, the percentages of SPCC and SPCL depletion might be underestimated.