Hexyl hexanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

8 May 2018 - 29 My 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:

05 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.

Test material

Test material information

Constituent 1

Reference substance name:

Hexyl hexanoate

EC Number:

228-952-4

EC Name:

Hexyl hexanoate

Cas Number:

6378-65-0

Molecular formula:

C12H24O2

IUPAC Name:

hexyl hexanoate

Test material form:

liquid

Specific details on test material used for the study:

4.1.1. Test Item (Hexyl Caproate)
Identification: Hexyl Caproate
Appearance: Clear colourless liquid
Batch: 202462
Purity/Composition: 99.29%
Test item storage: At room temperature
Stable under storage conditions until: 19 November 2019 (expiry date)
Additional information
Test Facility test item number: 209455/A
Purity/Composition correction factor: No correction factor required
Chemical name (IUPAC, synonym or trade name: Hexyl Hexanoate
CAS number: 6378-65-0
EC number: 228-952-4
Molecular structure:


Molecular formula: C12H24O2
Molecular weight: 200.32 g/mol
Specific gravity / density: 0.8606

In chemico test system

Details on the study design:

Objectives
The study was conducted to quantify the reactivity of the test material towards model synthetic peptides containing either lysine or cysteine. The data is 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 DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article. Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with UV detection. Cysteine and lysine peptide percent depletion (PPD) values were then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
Test Article Incubation
Each test solution was prepared at ratios of 1:10 and 1:50 with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25°C or 24±2 hours. At the end of the incubation period the samples were visually inspected for precipitate formation.

Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm or equivalent
Wavelength: 220 nm
Guard column: Phenomenex Security Guard c18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30°C
Sample temperature: 25°C
Injection volume: 5 µL

Mobile Phase:
Phase A: 0.1% (v/v) of trifluoroacetic acid in MilliQ water
Phase B: 0.085% (v/v) of trifluoroacetic acid in acetonitrile

Gradient: Time (min) Phase A Phase B
0 90 10
10 75 25
11 10 90
13 10 90
13.5 90 10
20 90 10

Reference and Co-elution Controls
Reference controls were prepared for each peptide.
Reference Control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
Reference Control C for cysteine was prepared by adding 750 µL of peptide stock solution to 200 µL of acetonitrile and 50 µL vehicle.
Reference Control C for lysine was prepared by adding 750 µL of peptide stock solution to 250 µL vehicle.
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that acetonitrile did not impact the percent peptide depletion.

Co-elution controls were prepared to detect possible co-elution of the test article with the peptides. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test article solution was used to detect possible co-elution of the test article with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test article solution was used to detect possible co-elution of the test article with lysine.

Calibration Curves for Peptides
Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
Standard 1 for cysteine was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
Standard 1 for lysine was prepared at approximatively 0.534 mM by dilution of 800 µL of the peptide stock solution (0.667 mM) with 200 µL of acetonitrile.
Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
Samples of dilution buffer alone were also prepared.

Sample Analysis Sequence
The analysis sequence for each peptide was as follows:
System suitability Standard 1 Dilution buffer
Calibration standards and reference controls Standard 1
Standard 2
Standard 3
Standard 4
Standard 5
Standard 6
Dilution Buffer
Reference Control A, rep 1
Reference Control A, rep 2
Reference Control A, rep 3
Co-elution controls Co-elution control for test article
Reference controls Reference Control B, rep 1
Reference Control B, rep 3
First set of replicates Reference Control C, rep 1
Positive Control, rep 1
Test sample, rep 1
Second set of replicates Reference Control C, rep 2
Positive Control, rep 2
Test sample, rep 2
Third set of replicates Reference Control C, rep3
Positive Control, rep 3
Test sample, rep 3
Reference controls Reference Control B, rep 4
Reference Control B, rep 5
Reference Control B, rep 6

Results and discussion

Positive control results:

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 63.9% ± 2.7%. 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

Parameter:

other: %SPCC depletion/mean

Value:

5.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Parameter:

other: %SPCL depletion/mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Parameter:

other: Mean %SpCC/SPCL depletion

Value:

2.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

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 item, were all within the acceptability criteria for the DPRA.

Any other information on results incl. tables

Solubility Assessment of the Test Item

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

Solubility of the 100 mM test item solution prepared in ACN, isopropanol, acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v) 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, the test item did not dissolve in the phosphate buffer solution. 

Solubility of the 100 mM test item solution prepared in ACN, isopropanol, acetone:ACN (1:1, v/v) and DMSO:ACN (1:9, v/v) 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, the test item did not dissolve in the ammonium acetate buffer solution.   

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

Cysteine Reactivity Assay

Two experiments were performed to determine the reactivity of Hexyl Caproate towards SPCC. For the first experiment performed on the 22^nd of May 2018, the results were not accepted since the mean of the Reference Control Samples A was below the acceptance criteria. The results of this experiment will be included in the raw data files of the study but will not be reported.

During the second experiment performed on the 28^th of May 2018, the reactivity ofHexyl Caproatetowards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 24 hours of incubation at 25±2.5°C. Representative chromatograms of CCcys-209455/Aand209455/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 inTable 3 (Appendix 3).

Acceptability of the Cysteine Reactivity Assay

The SPCC standard calibration curve is presented in Figure 1 (Appendix 2). The correlation coefficient (r²) of the SPCC standard calibration curve was 0.996. Since the r²was >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 0.517 ± 0.003 mM while the mean peptide concentration of Reference Controls C was 0.517 ± 0.007 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 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 1.0%. 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 17.95. The mean A₂₂₀/A₂₅₈ ratio± 10% range was16.16-19.75. 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 63.9% ± 2.7%. 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 forthe Test Item

Preparation of a 100 mMHexyl Caproatestock solution in ACN showed thatthe test itemwas 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 as well as after incubation a precipitate 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 the test material 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 209455/A-cys samples, the mean SPCC A₂₂₀/A₂₅₈ area ratio was 18.23. Since this was within the 16.16-19.75 range, this again indicated that there was no co‑elution ofthe test itemwith SPCC.

The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion forthe test itemwas 5.4%± 1.1%.

 Lysine Reactivity Assay

Two experiments were performed to determine the reactivity of Hexyl Caproate towards SPCL. For the first experiment performed on the 22^nd of May 2018, the results were not accepted since the means of the Reference Control Samples A and C were above the acceptance criteria. The results of this experiment will be included in the raw data files of the study but will not be reported.

During the second experiment performed on the 28^th of May 2018, the reactivity ofHexyl Caproatetowards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 24 hours of incubation at 25±2.5°C. Representative chromatograms of CClys-209455/Aand209455/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 0.997. 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 0.547 ± 0.002 mM while the mean peptide concentration of Reference Controls C was 0.537 ± 0.026 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 2.6%. 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 15.75. The mean A₂₂₀/A₂₅₈ratio± 10% range was 14.18-17.33. Each sample showing an A₂₂₀/A₂₅₈ratio 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 53.2% ± 2.6%. 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 mMHexyl Caproatestock 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 precipitate 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 inAppendix 4). This demonstrated that there was no co-elution of the test item with SPCL. For the 209455/A-lys samples, the mean SPCL A₂₂₀/A₂₅₈ area ratio was 15.96. Since this was within the 14.18-17.33 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 forthe Test Item was 0.0%± 0.0%.

 DPRA Prediction and Reactivity Classification

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate 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 5.4% 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 2.7% and as a result the test item was negative in the DPRA and was classified in the “no or minimal reactivityclass” 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 Caproate	5.4%	±1.1%	0.0%	±0.0%	2.7%	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 Caproate 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 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:

The objective of this study was to determine the reactivity of Hexyl Caproate towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test item 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 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 study procedures described in this report were based on the most recent OECD guideline.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. An overview of the obtained assay validation parameters is presented in the table below.

Acceptability of theDirect 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	0.996	>0.99	0.997
Mean peptide concentration RC-A samples (mM)	0.50 ± 0.05	0.517 ± 0.003	0.50 ± 0.05	0.547 ± 0.002
Mean peptide concentration RC-C samples (mM)	0.50 ± 0.05	0.517 ± 0.007	0.50 ± 0.05	0.537 ± 0.026
CV (%) for RC samples B and C	<15.0	1.0	<15.0	2.6
Mean peptide depletion cinnamic aldehyde (%)	60.8-100	63.9	40.2-69.0	53.2
SD of peptide depletion cinnamic aldehyde (%)	<14.9	2.7	<11.6	2.6
SD of peptide depletion for the test item (%)	<14.9	1.1	<11.6	0.0

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

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 item, were all within the acceptability criteria for the DPRA.

Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate 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 5.4% 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 2.7% and as a result the test item was considered to be negative in the DPRA and classified in the “no or minimal reactivityclass” 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 Caproate	5.4%	±1.1%	0.0%	±0.0%	2.7%	Negative: No or minimal reactivity

SD = Standard Deviation.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. Hexyl Caproate was negative in the DPRA and was classified in the “no or minimal reactivityclass” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation 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.