Hexyl butyrate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2018 to 03 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

04 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide 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.

Test material

Test material information

Constituent 1

Reference substance name:

Hexyl butyrate

EC Number:

220-136-6

EC Name:

Hexyl butyrate

Cas Number:

2639-63-6

Molecular formula:

C10H20O2

IUPAC Name:

hexyl butanoate

Test material form:

liquid

Specific details on test material used for the study:

Appearance: Clear colourless liquid
Purity/Composition: See Certificate of Analysis
Test item storage: At room temperature protected from light

In chemico test system

Details on the study design:

MATERIALS AND METHODS
Test Item and Reference Item
Test Item (Hexyl Butyrate)
Identification: Hexyl Butyrate
Appearance: Clear colourless liquid
Batch: 202509
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: 209412/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name: Hexyl butanoate
CAS number: 2639-63-6
EC number: 220-136-6
Molecular structure:
Molecular formula: C10H20O2
Molecular weight: 172.26 g/mol
For Certificate of Analysis see Appendix 6.

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 Structure:
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), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol, 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 32.17 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1868 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
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
Acetone HiPersolv, VWR international, Amsterdam, The Netherlands
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
Isopropanol LiChrosolv, Merck
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 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-209412/A 750 µL Phosphate buffer pH 7.5
200 µL ACN
50 µL 209412/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 3 209412/A-cys-1 to 750 µL Stock solution of 0.667 mM SPCC
209412/A 209412/A-cys-3 200 µL ACN
50 µL 209412/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-209412/A 750 µL Ammonium acetate buffer pH 10.2
250 µL 209412/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 3 209412/A-lys-1 to 750 µL Stock solution of 0.667 mM SPCL
209412/A 209412/A-lys-3 250 µL 209412/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 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. The samples that showed a phase separation 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.

Results and discussion

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 74.5% ± 0.9%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: SPCC depletion

Value:

0.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Parameter:

other: SPCL depletion

Value:

0.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

RESULTS

Solubility Assessment ofthe Test Item

At a concentration of 100 mM, Hexyl Butyrate was not soluble in MQ and ACN:MQ (1:1, v/v), but was soluble in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) and isopropanol.

Solubility of the 100 mM test item solution prepared in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) or isopropanol 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 ACN followed by vortex mixing. For all four solvents, formation ofa phase separation in the form of an oily top layer was observed.

Solubility of the 100 mM test item solution prepared in ACN, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v) or isopropanol 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 vortex mixing. For all four solvents, formation ofa phase separation in the form of an oily top layer was observed.

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

Cysteine Reactivity Assay

The reactivity of Hexyl Butyrate towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 24.5 hours of incubation at 25±2.5°C. Representative chromatograms of CCcys-209412/Aand209412/A‑cys samples are presented inAppendix 4. An overview of the retention time at 220 nm and peak areas at 220 nm and 258 nm are presented inTable 3(Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented inFigure 1(Appendix 2). The correlation coefficient (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 0.503 ± 0.005 mM while the mean peptide concentration of Reference Controls C was 0.508 ± 0.005 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 inTable 5(Appendix 3). The Coefficient of Variation (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 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 19.84. The mean A₂₂₀/A₂₅₈ ratio± 10% range was 17.85-21.82. 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 aldehyde was 74.5% ± 0.9%. 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 mMHexyl Butyratestock solution in ACN showed thatthe test itemwas 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 phase separation in the form of an oily top layer was observed in the co-elution control (CC) and 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 cysteine reactivity assay for thetest itemare presented inTable 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 the209412/A-cys samples, the mean SPCC A₂₂₀/A₂₅₈area ratio was 20.10. Since his was within the 17.85-21.82 range, this again indicated that there was no co-elution ofthe 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 0.6%± 0.6%.

Lysine Reactivity Assay

The reactivity of Hexyl Butyrate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 24.5 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-209412/Aand209412/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 inFigure 2(Appendix 2). The correlation coefficient (r²) of the SPCL standard calibration curve was 0.996. Since the r²was >0.99, the SPCL standard calibration curve was accepted.

The results of the Reference Control samples A and C are presented inTable 10 (Appendix 3). The mean peptide concentration of Reference Controls A was 0.505 ± 0.009 mM while the mean peptide concentration of Reference Controls C was 0.501 ± 0.010 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 dissolvethe test itemdid 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 1.5%. 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 inTable 12 (Appendix 3). The mean area ratio (A₂₂₀/A₂₅₈) of the Reference Control samples was 18.37. The mean A₂₂₀/A₂₅₈ ratio ± 10% range was 16.53-20.21. 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 54.8% ± 1.4%. 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 Butyratestock solution in ACN showed that thetest itemwas 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 phase separation in the form of an oily top layer was observed in the CC andthetest 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 forthe test itemare presented inTable 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 the209412/A-lys samples, the mean SPCL A₂₂₀/A₂₅₈ area ratio was 18.37. Since this was within the 16.53-20.21 range, this again indicated that there was no co-elution ofthe 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 0.4%± 0.5%.

DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a phase separation was observed.

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 0.6% SPCC depletion while in the lysine reactivity assay the test item showed 0.4% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.5% 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 forthe 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 Butyrate	0.6%	±0.6%	0.4%	±0.5%	0.5%	Negative: No or minimal reactivity

SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, this DPRA test is valid. Hexyl Butyrate 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 phase separation was observed after the incubation period for both SPCC and 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 Butyrate (EC 220-136-6) was found to not be classified as a skin sensitiser under Regulation EC 1272/2008.