Hexyl hexanoate

Endpoint summary

Description of key information

Studies conducted to internationally recognised testing guidelines with GLP certificaiton. QSAR results validated with suitable testing set and substance falls into applicability domain.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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
C(CCOC(CCCCC)=O)CCC

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See QMRF and QPRF attached.

5. APPLICABILITY DOMAIN
See QMRF and QPRF attached.

6. ADEQUACY OF THE RESULT
See QMRF and QPRF attached.

Qualifier:

no guideline followed

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

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 Hexanoate 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 2

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

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.

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

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

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%).

Parameter:

other: %SPCC depletion/mean

Value:

5.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Parameter:

other: %SPCL depletion/mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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.

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.

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. 

 

 

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

8 Jun 2018 - 29 Jun 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:

Identification: Hexyl Caproate
Appearance: Clear colourless liquid
Purity/Composition: 99.29%
Test item storage: At room temperature

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
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.03 and the EC1.5 22 µM.

Experiment 2
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 5.97 and the EC1.5 32 µM.

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.35

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.66

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Hexyl Caproate 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 Caproate 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 Caproate 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 Caproate. The Imax was 1.35 and therefore no EC1.5 could be calculated.

Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• Hexyl Caproate 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.
• A luminescence activity induction was observed after treatment with Hexyl Caproate. The Imax was 1.66 and the EC1.5 1575 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 (22 µM and 32 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (4.03-fold and 5.97-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% (7.8% and 8.5% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Hexyl Caproate showed no toxicity (no IC30 and IC50 value). In the first experiment no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations, leading to an individual run conclusion of negative. In the second experiment an induction of the luciferase activity (EC1.5 value 1574 µM) was measured. Since the EC1.5 value was higher than 1000 µM the individual run conclusion of the second experiment was negative. The maximum luciferase activity induction (Imax) was 1.35-fold and 1.66-fold in experiment 1 and 2 respectively. Hexyl Caproate is classified as negative in the KeratinoSensTM assay since negative run conclusions were obtained in both experiments.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Hexyl Caproate is classified as negative (no biologically relevant 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 Caproate 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 202462 of Hexyl Caproate was a clear colourless liquid. The test item 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 (22 µM and 32 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (4.03-fold and 5.97-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% (7.8% and 8.5% in experiment 1 and 2, respectively).

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

Hexyl Caproate showed no toxicity (no IC30 and IC50 value). In the first experiment no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations, leading to an individual run conclusion of negative. In the second experiment an induction of the luciferase activity (EC1.5 value 1574 µM) was measured. Since the EC1.5 value was higher than 1000 µM the individual run conclusion of the second experiment was negative. The maximum luciferase activity induction (Imax) was 1.35-fold and 1.66-fold in experiment 1 and 2 respectively. Hexyl Caproate is classified as negative in the KeratinoSensTM assay since negative run conclusions were obtained in both experiments.

In conclusion, Hexyl Caproate is classified as negative (no biologically relevant 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 study available

Additional information:

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

A valid DPRA test was performed according to OECD TG 442C and GLP principles. For the DPRA assay Hexyl Caproate was dissolved in acetonitrile at 100 mM. Upon preparation as well as after incubation of the SPCC and SPCL test item samples, a precipitate was observed.

No co-elution of the test item with SPCC or SPCL was observed. 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 reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

A valid KeratinoSensTM assay was performed according to OECD 442D and GLP. For the KeratinoSensTM assay Hexyl Caproate was dissolved in DMSO to a final concentration of 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 which passed the acceptance criteria. Hexyl Caproate showed no toxicity (no IC30 and IC50 value). In the first experiment no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations, leading to an individual run conclusion of negative. In the second experiment an induction of the luciferase activity (EC1.5 value 1574 μM) was measured. Since the EC1.5 value was higher than 1000 μM the individual run conclusion of the second experiment was negative. The maximum luciferase activity induction (Imax) was 1.35-fold and 1.66-fold in experiment 1 and 2, respectively. Hexyl Caproate is classified as negative in the KeratinoSensTM assay since

negative run conclusions were obtained in both experiments.

According to DEREK NEXUS version 6.0.1, Hexyl Caproate is predicted to be not sensitizing to the skin and was shown not to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) according to the results of a

KeratinoSensTM assay. Hexyl Caproate showed minimal or no binding to cysteine and lysine moieties in the DPRA assay. It is noted that precipitation was observed after the incubation period for both SPCC and SPCL, which implies that potentially less test item remained in the solution to react with the peptide. Consequently, this negative result is uncertain and should be interpreted with caution.

Based on the current experimental data-set it is concluded that there are no indications that Hexyl Caproate 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

The available data on the registered substance is not sufficient to fulfill the criteria for classification as a skin sensitiser in accordance with the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).