Esterification products of salicylic acid and C13-(branched) alcohols

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 December 2017 to 2 Marhc 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:

tridecyl 2-hydroxybenzoate

EC Number:

947-975-7

Molecular formula:

C20H32O3

IUPAC Name:

tridecyl 2-hydroxybenzoate

Specific details on test material used for the study:

The test article, a clear liquid, was identified as Dermol TDSA (chemical name Tridecyl Salicylate) and was received at Covance on 31 October 2017 as follows:
Test Article Dermol TDSA
CAS Number 19666-16-1
Storage 15 to 25°C, protected from light
Batch Number P7202
Expiration Date October 2019
Purity 100%

A certificate of analysis for the test article was provided by the Sponsor and is presented in the Certificates of Analysis.

The test article was dissolved in acetonitrile. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.

In chemico test system

Details on the study design:

Objectives
The study was conducted to quantify the reactivity of Dermol TDSA 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 Formulation
The test article was dissolved in acetonitrile. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.

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.

Positive control
Cinnamaldehyde (CAS No. 104-55-2, batch number MKBT8955V, purity 99.1%, expiry 29 February 2020) was used as the positive control. The positive control was dissolved in acetonitrile at a concentration of 100 mM.

Peptides
The peptides, cysteine (lot number P170608-LC180433, purity 96.07%) and lysine (lot number P170608-LC107617, purity 96.32%) were obtained from RS Synthesis, Louisville, Kentucky, USA.

Stock solution
A stock solution containing cysteine at approximately 0.667 mM was prepared in 100 mM Phosphate Buffer pH 7.5 and a stock solution containing lysine at approximately 0.667 mM was prepared in 100 mM ammonium acetate buffer pH 10.2.
Formulations were prepared shortly before testing.

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 (see Protocol Deviations)

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).
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:

See "Any other information on results incl. tables"

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: Cysteine depletion value (%)

Value:

12.48

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Considered to be negative in the Direct Peptide Reactivity Assay

Results 2

Key result

Parameter:

other: Lysine depletion (%)

Value:

0.15

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: considered to be negative in the Direct Peptide Reactivity Assay

Other effects / acceptance of results:

Protocol Deviations

Procedure
Analytical Method - Injection Volume

Protocol Deviations
Analysis batch was run with a reduced injection volume of 5 µL instead of the 7 µL stated in the study protocol. This reduced injection volume was pre-approved by the study director prior to running the batch. There was no impact on integrity or outcome of the study as the reduced injection volume has been shown to improve integration of the peak.

These study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.

Any other information on results incl. tables

Protocol Deviations

Procedure

Analytical Method  - Injection Volume

Protocol Deviations

Analysis batch was run with a reduced injection volume of 5 µL instead of the 7 µL stated in the study protocol. This reduced injection volume was pre-approved by the study director prior to running the batch. There was no impact on integrity or outcome of the study as the reduced injection volume has been shown to improve integration of the peak.

These study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.

Cysteine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C	PPD	Mean PPD	SD
		Mean Peptide Peak Area
Test Article	12.29	13.36	8.01	12.5	4.7
	11.05		17.3
	11.74		12.1
Positive Control	4.57	13.36	65.8	64.7	1.1
	4.85		63.7
	4.72		64.7

The r²value for the standard calibration curve was 0.99617.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.52	0.49	0.47	0.5
C	0.51	0.46	0.47	0.48

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	15.09
	2	13.6
	3	13.67
	4	12.85
	5	11.91
	6	12.96
C	1	14.28
	2	12.8
	3	12.99
Mean		13.35
SD		0.93
CV		6.99

Lysine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C	PPD	Mean PPD	SD
		Mean Peptide Peak Area
Test Article	25.77	23.97	0	0.15	0.26
	23.86		0.46
	24.08		0
Positive Control	9.77	23.97	59.24	56.28	3.05
	10.44		56.45
	11.23		53.15

The r²value for the standard calibration curve was 0.99997.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.5	0.5	0.5	0.5
C	0.5	0.5	0.51	0.5

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	24.72
	2	23.91
	3	23.9
	4	23.79
	5	24.49
	6	23.87
C	1	23.81
	2	23.83
	3	24.26
Mean		24.06
SD		0.34
CV		1.42

Applicant's summary and conclusion

Interpretation of results:

other: The test article, Dermol TDSA, was considered to be negative in the Direct Peptide Reactivity Assay.

Conclusions:

The cysteine depletion value was 12.48%, the lysine depletion value was 0.15% and the mean of the cysteine and lysine depletion values was therefore 6.31%.
The test article, Dermol TDSA, was considered to be negative in the Direct Peptide Reactivity Assay.

Executive summary:

The study was conducted to quantify the reactivity of Dermol TDSA towards model synthetic peptides containing either lysine or cysteine. 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 test article was dissolved in acetonitrile at a concentration of 100 mM.

The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light and set at 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The cysteine depletion value was 12.48%, the lysine depletion value was 0.15% and the mean of the cysteine and lysine depletion values was 6.31%.

The test article,Dermol TDSA, was considered to be negativein the Direct Peptide Reactivity Assay.