Hexyl propionate

Endpoint summary

Description of key information

Study carried out according to recognised testing guidelines with GLP certification.

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:

weight of evidence

Study period:

03 May 2018 to 08 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

04 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Pepditde reactivity Assay - DPRA

Justification for non-LLNA method:

This is a Non in vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.

Specific details on test material used for the study:

Appearance: Clear colourless liquid
Test item storage: At room temperature protected from light

Details on the study design:

INTRODUCTION
The objective of this study was to determine the reactivity of Hexyl Propionate towards model synthetic peptides containing either cysteine or lysine, and to categorize the test item in
one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

Background
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25°C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

The design of this study is based on the following study guideline:
• OECD Guideline for the Testing of Chemicals, Guideline 442C. In Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA) (4 February 2015)

The Study Director signed the study plan on 16 Apr 2018. The experimental start date was 03 May 2018, and the experimental completion date was 08 May 2018.

MATERIALS AND METHODS:
Test Item and Reference Item:
Test Item (Hexyl Propionate)
Identification: Hexyl Propionate
Appearance: Clear colourless liquid
Batch: 601156
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 16 September 2018 (expiry date)

Additional information:
Test Facility Test Item Number: 209413/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or
trade name: Hexyl propanoate
CAS number: 2445-76-3
EC number: 219-495-1
Molecular formula: C9H18O2
Molecular weight: 158.24 g/mol

Reference Item (Positive Control Cinnamic Aldehyde)
Identification: Cinnamic aldehyde
Test Facility Test Item Number: RS473/A
Appearance: Yellow liquid
CAS Number: 104-55-2
Molecular Formula: C9H8O
Molecular Weight: 132.16 g/mol
Batch: MKBP1014V
Purity: 98.4%
Test item storage: In the refrigerator (2-8°C) Stable under storage conditions until: 31 May 2018
Supplier: Sigma-Aldrich Chemie GmbH, Steinheim, Germany
Purity/composition correction factor: Yes
For Certificate of Analysis see Appendix 6.

Test Item Characterization:
The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis was provided to the Test Facility and is presented in Appendix 6.

Reserve Samples:
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

Test Item Inventory and Disposition:
Records of the receipt, distribution, and storage of test item were maintained. With the exception of reserve samples, all unused Sponsor-supplied test item will be discarded or returned to the Sponsor after completion of the scheduled program of work. Records of the decisions made will be kept at the Test Facility.

Dose Formulation and Analysis:
Preparation of Test Item:
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 not cloudy nor have noticeable precipitate. The following solvents were evaluated: acetonitrile (ACN), isopropanol, dimethylsulfoxide (DMSO):ACN (1:9, v/v), methanol (MeOH) and ethanol (EtOH).

Test item stock solutions were prepared freshly for each reactivity assay.

For both the cysteine and lysine reactivity assay 29.88 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1888 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.

Any residual volumes were discarded.

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.
Batch: See Appendix 7 for detailed information.
Storage:The peptides were stored in the freezer (≤-15°C) for a maximum of 6 months.

Reagents:
Acetonitrile (ACN): HPLC grade, Fisher Chemicals, Loughborough, England
Ammonium acetate: Fractopur, Merck, Darmstadt, Germany
Ammonium hydroxide: 25%, Merck
Dimethylsulfoxide (DMSO): Seccosolv, Merck
Disodium hydrogen phosphate (Na2HPO4·12H2O): Emsure, Merck
Ethanol (EtOH): ACS ISO, Merck
Isopropanol: LiChrosolv, Merck
Methanol (MeOH): BioSolve, Valkenswaard, The Netherlands
MilliQ-water (MQ): Tap water purified by reversed osmosis and subsequently passed over activated carbon and ion exchange cartridges; Millipore, Bedford, MA, USA
Sodium dihydrogenphosphate dehydrate (NaH2PO4·H2O): Emsure, Merck
Trifluoroacetic acid (TFA): >99% Sigma Aldrich, Zwijndrecht, The Netherlands

Experimental Design:
Preparation of Solutions for Cysteine Reactivity Assay:
Synthetic Peptide Containing Cysteine (SPCC) Stock Solution:
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions:
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

SPCC Calibration Curve:
A SPCC calibration curve was prepared as described in the table below.


Preparation of SPCC Calibration Curve:
SPCC calibration solutions SPCC concentration (mM) Preparation
STDcys1 0.534 1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN
STDcys2 0.267 1 mL STDcys1 + 1 mL STDcys7
STDcys3 0.133 1 mL STDcys2 + 1 mL STDcys7
STDcys4 0.067 1 mL STDcys3 + 1 mL STDcys7
STDcys5 0.033 1 mL STDcys4 + 1 mL STDcys7
STDcys6 0.017 1 mL STDcys5 + 1 mL STDcys7
STDcys7 0 0 8 mL phosphate buffer (pH 7.5) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples:
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.

Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CCcys-209413/A 750 µL Phosphate buffer pH 7.5
200 µL ACN
50 µL 209413/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PCcys-1 to PCcys-3 750 µL Stock solution of 0.667 mM SPCC
200 µL ACN
50 µL Cinnamic aldehyde solution (100 mM in ACN)

Test item 209413/A 3 209413/A-cys-1 to 750 µL Stock solution of 0.667 mM SPCC
209413/A-cys-3 200 µL ACN
50 µL 209413/A test solution (100 mM)

Preparation of Solutions for Lysine Reactivity Assay:
Synthetic Peptide Containing Lysine (SPCL) Stock Solution:
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions:
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve:
A SPCL peptide calibration curve was prepared as described in the table below.

Preparation of SPCC Calibration Curve

SPCC calibration solutions SPCC concentration (mM) Preparation
STDcys1 0.534 1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN
STDcys2 0.267 1 mL STDcys1 + 1 mL STDcys7
STDcys3 0.133 1 mL STDcys2 + 1 mL STDcys7
STDcys4 0.067 1 mL STDcys3 + 1 mL STDcys7
STDcys5 0.033 1 mL STDcys4 + 1 mL STDcys7
STDcys6 0.017 1 mL STDcys5 + 1 mL STDcys7
STDcys7 0 8 mL phosphate buffer (pH 7.5) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in the table below.

Preparation of Co-elution Control, Test Item and Positive Control Samples

Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CCcys-209413/A 750 µL Phosphate buffer pH 7.5
200 µL ACN
50 µL 209413/A test solution (100 mM)

Cinnamic aldehyde (PC) 3 PCcys-1 to PCcys-3 750 µL Stock solution of 0.667 mM SPCC
200 µL ACN
50 µL Cinnamic aldehyde solution
(100 mM in ACN)
Test item 209413/A 3 209413/A-cys-1 to 750 µL Stock solution of 0.667 mM SPCC
209413/A-cys-3 200 µL ACN
50 µL 209413/A test solution (100 mM)


Preparation of Solutions for Lysine Reactivity Assay:
Synthetic Peptide Containing Lysine (SPCL) Stock Solution:
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions:
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve:
A SPCL peptide calibration curve was prepared as described in the table below.

Preparation of SPCL Calibration Curve
SPCL calibration solutions SPCL concentration (mM) Preparation
STDlys1 0.534 1600 µL stock solution of 0.667 mM SPCL + 400 µL ACN
STDlys2 0.267 1 mL STDlys1 + 1 mL STDlys7
STDlys3 0.133 1 mL STDlys2 + 1 mL STDlys7
STDlys4 0.067 1 mL STDlys3 + 1 mL STDlys7
STDlys5 0.033 1 mL STDlys4 + 1 mL STDlys7
STDlys6 0.017 1 mL STDlys5 + 1 mL STDlys7
STDlys7 0 8 mL ammonium acetate buffer (pH 10.2) + 2 mL ACN

Co-elution Control, Test Item and Positive Control Samples
The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive
control samples (PC) were prepared as described in the table below.

Preparation of Co-elution Control, Test Item and Positive Control Samples
Sample Replicates Sample code Preparation
Co-elution control (CC) 1 CClys-209413/A 750 µL Ammonium acetate buffer pH 10.2
250 µL 209413/A test solution (100 mM)
Cinnamic aldehyde (PC) 3 PClys-1 to PClys-3 750 µL Stock solution of 0.667 mM SPCL
250 µL Cinnamic aldehyde solution
(100 mM in ACN)
Test item 209413/A 3 209413/A-lys-1 to 750 µL Stock solution of 0.667 mM SPCL
209413/A-lys-3 250 µL 209413/A test solution (100 mM)

Sample Incubations:
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 23 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.

HPLC-PDA Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC-PDA. Sample analysis was performed using the following systems:
System 1 (used for Cysteine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
• LC Column oven 300 (Thermo Scientific)
• Surveyor PDA detector (Thermo Scientific)
System 2 (used for Lysine Reactivity Assay):
• Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
• HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
• Column Oven #151006 (Grace, Worms, Germany)

• Surveyor PDA detector (Thermo Scientific)
All samples were analyzed according to the HPLC-PDA method presented in Table 1 (Appendix 1). The HPLC sequences of the cysteine and lysine reactivity assay for the test item are presented in Table 2 (Appendix 1).

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.

Positive control results:

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 70.0% ± 1.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Key result

Parameter:

other: SPCC depletion

Value:

0.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Parameter:

other: SPCL depletion

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Solubility Assessment of the Test Item

At a concentration of 100 mM, Hexyl Propionate was soluble in ACN, DMSO:ACN (1:9,v/v), isopropanol, EtOH and MeOH.

Solubility of the 100 mM test item solution prepared in ACN, DMSO:ACN (1:9, v/v),isopropanol, EtOH or MeOH was investigated in the SPCC assay buffer by mixing 50 µL of the 100 mM test item solution with 750 µL phosphate buffer pH 7.5 and 200 µL ACNfollowed by vortex mixing. For ACN, EtOH and MeOH formation of a precipitate in theform of small particles was observed, for DMSO:ACN (1:9,v/v) formation of a suspensionwas observed while for isopropanol the test item also did not dissolve.

Solubility of the 100 mM test item solution prepared in ACN, DMSO:ACN (1:9,v/v),isopropanol, EtOH or MeOH was investigated in the SPCL assay buffer by mixing 250 µL of the 100 mM test item solution with ammonium acetate buffer pH 10.2 followed by vortexmixing. For all ACN, DMSO:ACN (1:9,v/v), EtOH and MeOH formation of a precipitate inthe form of oily droplets was observed while for isopropanol the test item also did not dissolve.

As ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test itemin this DPRA study.

Cysteine Reactivity Assay

The reactivity of Hexyl Propionate towards SPCC was determined by quantification of theremaining concentration of SPCC using HPLC-PDA analysis, following 23 hours of incubation at 25±2.5°C. Representative chromatograms of CCcys-209413/A and 209413/A-cys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 3 (Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlationcoefficient (r2) of the SPCC standard calibration curve was 0.998. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented in Table 4 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.505 ± 0.003 mM while the

mean peptide concentration of Reference Controls C was 0.506 ± 0.006 mM. The means of Reference Control samples A and C were both within the acceptance criteria of

0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The SPCC peak areas for Reference controls B and C are presented in Table 5 (Appendix 3). The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.4%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCC A220/A258 area ratios of Reference controls A, B and C are presented in Table 6 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 19.64. The mean A220/A258 ratio ± 10% range was 17.68-21.60. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 7 (Appendix 3). The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehydewas 70.0% ± 1.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

Results Cysteine Reactivity Assay for the Test Item

Preparation of a 100 mM Hexyl Propionate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test item samples were visually inspected. Upon preparation, a precipitate was observed in the CC and test item samples. After incubation no precipitate or phase separation was observed in any of the samples.

The results of the cysteine reactivity assay for the test item are presented in Table 8 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCC (see chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCC. For the 209413/A-cys samples, the mean SPCC A220/A258 area ratio was

19.86. Since this was within the 17.68-21.60 range, this again indicated that there was no co-elution of the test item with SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test item was 0.2% ± 0.4%.

Lysine Reactivity Assay

The reactivity of Hexyl Propionate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 23 hours of

incubation at 25±2.5°C. Representative chromatograms of CClys-209413/A and 209413/A-lys samples are presented in Appendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented in Table 9 (Appendix 3).

Acceptability of the Lysine Reactivity Assay

The SPCL standard calibration curve is presented in Figure 2 (Appendix 2). The correlation coefficient (r2) of the SPCL standard calibration curve was 0.998. Since the r2 was >0.99, the SPCL standard calibration curve was accepted. The results of the Reference Control samples A and C are presented in Table 10 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.493 ± 0.002 mM while the mean peptide concentration of Reference Controls C was 0.494 ± 0.004 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The SPCL peak areas for Reference controls B and C are presented in Table 11 (Appendix 3). The CV of the peptide areas for the nine Reference Controls B and C was 1.3%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The SPCL A220/A258 area ratios of Reference controls A, B and C are presented in Table 12 (Appendix 3). The mean area ratio (A220/A258) of the Reference Control samples was 18.32. The mean A220/A258 ratio ± 10% range was 16.48-20.15. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

The results of the positive control cinnamic aldehyde are presented in Table 13 (Appendix 3). The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference

Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 52.6% ± 1.2%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).

Results Lysine Reactivity Assay for the Test Item

Preparation of a 100 mM Hexyl Propionate stock solution in ACN showed that the test item was dissolved completely. Upon preparation and after incubation, both the CC as well as the test item samples were visually inspected. Upon preparation as well as after incubation a precipitate was observed in the CC and the test item samples. In this case one cannot be sure how much test item remained in the solution to react with the peptide.

The results of the lysine reactivity assay for the test item are presented in Table 14 (Appendix 3). In the CC sample no peak was observed at the retention time of SPCL (see

chromatogram in Appendix 4). This demonstrated that there was no co-elution of the test item with SPCL. For the 209413/A-lys samples, the mean SPCL A220/A258 area ratio was

18.53. Since this was within the 16.48-20.15 range, this again indicated that there was no co-elution of the test item with SPCL.

The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Item was 0.0% ± 0.0%.

DPRA Prediction and Reactivity Classification

Upon preparation of the SPCC and SPCL test item samples, a precipitate was observed. After incubation, a phase separation was only observed in the SPCL test item samples.

An overview of the individual results of the cysteine and lysine reactivity assays as well as the mean of the SPCC and SPCL depletion are presented in the table below. In the cysteine

reactivity assay the test item showed 0.2% 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

0.1% and as a result the test item 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.

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
Hexyl Propionate	0.2%	±0.4%	0.0%	±0.0%	0.1%	Negative: No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, this DPRA test is valid. Hexyl Propionate 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. However, since precipitation was observed after the incubation period for SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Executive summary:

In this guideline (OECD 442C) study, performed with GLP certification, the test substance Hexyl Propionate (EC 219-495-1) was found to not be classified as a skin sensitiser under Regulation EC 1272/2008.

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

QSAR prediction

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
DEREK NEXUS v6.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
SMILES: CCC(OCCCCCC)=O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See QMRF & QPRF below.

5. APPLICABILITY DOMAIN
See QMRF & QPRF below.

6. ADEQUACY OF THE RESULT
See QMRF & QPRF below.

Qualifier:

no guideline required

Principles of method if other than guideline:

Derek Nexus v6.0 contains 90 alerts for skin sensitisation, together with reasoning rules encoding physicochemical descriptors. In addition to a prediction of skin sensitisation potency for alerting query compounds, Derek evaluates potentially misclassified and unclassified features in compounds that do not activate skin sensitisation alerts or examples.

GLP compliance:

no

Remarks:

Full records of the QSAR model and input data are recorded in the QMRF and QPRF.

Justification for non-LLNA method:

This is a Non in vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.

Specific details on test material used for the study:

Not applicable to QSAR.

Positive control results:

Not applicable to QSAR.

Parameter:

other: Alerts

Value:

0

Other effects / acceptance of results:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. Hexyl Propionate is predicted to be not sensitizing to the skin.

Interpretation of results:

GHS criteria not met

Conclusions:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for the test item.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 May 2018 to 08 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This is a Non in vivo test and the test material is used in cosmetic ingredients. Regulation 1223/2009 Article 18 restricts the use of in vivo studies on these types of raw materials.

Specific details on test material used for the study:

Test item information
Identification: Hexyl Propionate
Appearance: Clear colourless liquid
Batch: 601156
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light
Stable under storage conditions until: 16 September 2018 (expiry date)
Additional information
Test Facility test item number: 209413/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Chemical name (IUPAC, synonym or
trade name:

Hexyl propanoate

CAS number: 2445-76-3
EC number: 219-495-1
Molecular weight: 158.24

Details on the study design:

The study was conducted to investigate the potential of the test material to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by
luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Specifications
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland. Identification
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative control.

Preparation of Cultures
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.

Treatment Plate Preparation
The cells were 80-90% confluent (see Section 9 for details of protocol deviations). On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37±1°C, 5% (v/v) CO2, for 24±1 hours.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Each plate was sealed and incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at two independent repetitions each containing three replicates of each concentration.
The data for repetition 1 was obtained from a repeat experiment as the initial experiment did not meet the acceptance criteria for the positive or negative controls.
The data from the initial experiment has not been reported.
Discordant results were obtained between the two repetitions, therefore a third repetition containing three replicates was performed.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different passages.

Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2.
The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight.
After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate
reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Positive control results:

Experiment 1 and 2
Concentration (µM) 7.8 16 31 63 125 250
Exp 1 luminescence 0.91 0.95 1.05 1.15 1.73*** 2.81***
Exp 1 viability (%) 96.9 103.1 107.3 112.1 118.5 123.0
Exp 2 luminescence 1.06 1.21 1.18 1.57*** 1.72*** 2.25***
Exp 2 viability (%) 94.6 86.8 94.7 90.3 90.6 103.1
*** p<0.001 Student’s t test

Run / experiment:

other: 1

Parameter:

other: Maximum luciferase activity (IMax fold)

Value:

1.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: 2

Parameter:

other: Maximum luciferase activity (Imax fold)

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Hexyl Propionate was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway. An overview of the viability and luciferase
activity induction is summarized in Table 1 and Figure 2-3. The results of the positive control are summarized in Table 2 and Figure 4-5. An overview of EC1.5, Imax, IC30 and IC50
values is given in Table 3. The individual raw data are presented in Appendix 3 and Appendix 4. The historical control data are presented in Appendix 5.
Two independent experiments were performed. The cells were in these experiments incubated with Hexyl Propionate in a concentration range of 0.98 – 2000 µM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

Experiment 1
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• Hexyl Propionate showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Hexyl Propionate. The Imax was 1.06 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.81 and the EC1.5 100 µM.

Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• Hexyl Propionate showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with Hexyl Propionate. The Imax was 1.05 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.25 and the EC1.5 57 µM.
Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant 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 (100 µM and 57 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.81-fold and 2.25-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.2% and 7.0% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

Hexyl Propionate showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.06-fold and 1.05-fold in experiment 1 and 2 respectively. Hexyl Propionate is classified as
negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.
In conclusion, Hexyl Propionate 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.

Executive summary:

The objective of this study was to evaluate the ability of Hexyl Propionate to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

The study procedures described in this report were based on the most recent OECD guideline.

Batch 601156 of Hexyl Propionate was a clear colourless liquid. Hexyl Propionate was dissolved in dimethyl sulfoxide at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant 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 (100 µM and 57 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.81-fold and 2.25-fold in experiment 1 and 2, respectively).

• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.2% and 7.0% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Hexyl Propionate showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.06-fold and 1.05-fold in experiment 1 and 2 respectively. Hexyl Propionate is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

In conclusion, Hexyl Propionate 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)

Additional information:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization for hexyl propionate. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. Hexyl Propionate is predicted to be not sensitizing to the skin.

 

A valid Direct Peptide Reactivity Assay (DPRA) test was performed according to OECD 442C and GLP. Acetonitrile was found to be an appropriate solvent to dissolve the test item and was therefore used in this study. The test item was dissolved completely at 100 mM. Upon preparation of the test item samples with synthetic peptides containing either cysteine (SPCC) or lysine (SPCL), a precipitate was observed. After incubation, a phase separation was only observed in the SPCL test item samples. In this case it is not clear how much test item remained in the solution to react with the peptide. No co-elution of hexyl propionate with SPCC or SPCL was observed. In the cysteine reactivity assay the test item showed 0.2% 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 0.1% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

A valid KeratinoSens^TM assay was performed according to OECD 442D and GLP. Hexyl propionate was dissolved in dimethyl sulfoxide at 200 mM, the final test concentrations were 0.98 – 2000 µM (2-fold dilution series). No precipitate was observed at any dose level tested. Two independent experiments were performed. Hexyl propionate showed no toxicity (no IC₃₀ and IC₅₀ value) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.06-fold and 1.05-fold in experiment 1 and 2 respectively. Hexyl propionate is classified as negative in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations up to and including 2000 µM (highest dose required in the current guideline).

According to DEREK NEXUS version 6.0.1, hexyl propionate is predicted to be not sensitizing to the skin. Hexyl propionate showed minimal or no binding to cysteine and lysine moieties in the DPRA assay. It is noted that precipitation was seen in the lysine reactivity assay, which implies that potentially less test item remained in the solution to react with the peptide. However, the mean percent SPCL depletion of 0.1% indicates that no or only very limited binding of the test item took place with lysine. However, since precipitation was observed after the incubation period for SPCL, one cannot be sure how much test item remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care. Based on these results hexyl propionate is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Furthermore, hexyl propionate was shown not to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) according to the results of a KeratinoSens^TM assay.

Based on the current experimental data-set it is concluded that there are no indications that hexyl propionate has skin sensitizing properties, although the precipitation in the DPRA may underestimate the actual binding to protein moieties. Taking into account that the substance has been concluded to be safe for use as a food additive (and has been used as such for several decades), it is concluded that the substance does not have to be classified for skin sensitizing properties.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the weight of evidence data presented, the registered substance does not fulfill the criteria for classification as a skin sensitiser in accordance with the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).