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

Endpoint summary

Description of key information

The key studies were conducted to internationaly recognised testing guidelines and with GLP certification.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 December 2017 to 2 Marhc 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:

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.

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

Positive control results:

See "Any other information on results incl. tables"

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

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.

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

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.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 January 2018 to 3 April 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.

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.

Test Article Formulation
Test formulations were prepared using DMF. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 200 mM.
Serial dilutions were made (from the 200 mM stock) using the vehicle to obtain 12 master concentrations (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).
The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 0.98 to 2000 µM.
Formulations were prepared shortly before testing. In Experiment 1, the stock formulation was mixed using a vortex mixer.

Negative and Positive Control Article Formulation
The negative control was diluted into culture medium containing serum so that the final concentration was 1%.
The positive control was prepared at a concentration of 6.4 mM in DMSO. Five master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 6.4 mM stock solution). The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 4 to 64 µM.

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.

Key result

Parameter:

other: ARE-Nrf2 Luciferase Test

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Negative result

Calculation of I_maxand EC_1.5Values

Luminescence readings and fold increases are given in Table 9.1 (attached) andTable 9.2 (attached)

The overall maximal fold increase (I_max) for Experiment 1 was 2.17. However, this was not statistically significant, therefore there was no EC_1.5value.

The I_maxfor Experiment 2 was 1.62 and the EC_1.5value was 28.53.

Viability

MTT-absorbance readings are given inTable 9.3 (attached).

The cell viability at the EC_1.5determining concentration was 31.23 % in

Experiment 2. The cell viability measurement was not applicable in Experiment 1 as there was no EC_1.5determining concentration.

The IC₅₀values were 26.21 and 25.02µM in Experiments 1 and 2, respectively.

The IC₃₀values were 21.79 and 18.39µM in Experiments 1 and 2, respectively.

Assay Acceptance

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 4 to 64 µM in Experiment 1 and at concentrations of 8 to 64 µM in Experiment 2.

The EC_1.5values for the positive control were 3.13 and 6.49 µM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 µM were 3.11 and 17.34 in Experiments 1 and 2, respectively.

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 17.17% and 12.15% in Experiments 1 and 2, respectively.

The acceptance criteria were met in Experiment 1 with the exception that the EC_1.5for the positive control was below the range specified in the protocol (7 to 30µM).

The acceptance criteria were met in Experiment 2 with the exception that the EC_1.5for the positive control was below the range specified in the protocol (7 to 30µM) and the average induction at 64 µM was above the range specified in the protocol (2 to 8).

However, in both experiments luciferase induction values for the positive control showed a clear dose response, therefore this was considered a valid experiment.

Conclusions:

The Imax in Experiment 1 was not statistically significant when compared to the negative control.
Although the Imax in Experiment 2 was statistically significant when compared to the negative control, the cell viability at the lowest concentration with induction of luciferase activity above 1.5-fold was less than 70% and no dose response was seen.
All four conditions required for a positive prediction were therefore not met in either experiment. The test article, Dermol TDSA, was considered to be negative in the ARE-Nrf2 Luciferase Test.

Executive summary:

The study was conducted to investigate the potential of Dermol TDSA 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 test article was dissolved in dimethylformamide (DMF) to the final stock concentration (200 mM). Serial dilutions were then made using DMF to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).

The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.98 to 2000 µM.

Aliquots of 50 µL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO₂in air, in a humidified environment for 48±1 hours.

After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO₂. The MTT medium was then 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) CO₂in 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.

After the 48-hour exposure period, the cells in the luciferase plates 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.

The results are summarised as follows:

Criteria	Experiment 1	Experiment 2
Imax	2.17 (but not statistically significant)	1.62 (and statistically significant)
Cell Viability	Not applicable	31.23%
EC1.5	>2000 µM	28.53 µM
Dose Response	Yes	No

The acceptance criteria were met in Experiment 1 with the exception that the EC_1.5for the positive control was below the range specified in the protocol at 3.13µM.

The acceptance criteria were met in Experiment 2 with the exception that the EC_1.5for the positive control was below the range specified in the protocol at 6.49µM and the average induction at 64 µM was above the range specified in the protocol at 17.34µM.

However, in both experiments luciferase induction values for the positive control showed a clear dose response, therefore this was considered a valid experiment.

The I_maxin Experiment 1 was not statistically significant when compared to the negative control.

Although the I_maxin Experiment 2 was statistically significant when compared to the negative control, the cell viability at the lowest concentration with induction of luciferase activity above 1.5-fold was less than 70% and no dose response was seen.

All four conditions required for a positive prediction were therefore not met in either experiment. The test article, Dermol TDSA, was considered to be negative in the ARE-Nrf2 Luciferase Test.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 2018 - 23 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E The human Cell Line Activation Test (h-CLAT)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

This study is an in vitro alternative used to avoid the requirement to use in vivo studies to investigate this end point

Specific details on test material used for the study:

The test article, a clear liquid, was identified as Tridecyl Salicylate and was received at the test house as follows:
- Test Article - Isodecyl 3,5,5-trimethylhexanoate
- Purity - UVCB 100%

Details of test system:

THP-1 cell line [442E]

Details on the study design:

-Specifications
Human monocytic leukemia cell line, THP-1 (an immortalised human monocytic leukemia cell line, used as a surrogate for dendritic cells), supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.
-Identification
The test system was suitably labelled to clearly identify the study number, test article, test article concentration, positive and vehicle controls.

-Cell Culture Maintenance
THP-1 cells were cultured, at 37ºC under 5% CO2 and humidified atmosphere, in RPMI0-1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin.
The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 106 cells/mL and maintained at a density from 0.1 x 106 to 0.8 x 106 cells/mL. Cell density did not exceed 1 x 106 cells/mL.

-Reactivity Check
This was performed using DNCB (CAS no. 97-00-7, ≥99% purity), nickel sulphate (CAS no. 10101-97-0, 99% purity) and lactic acid (CAS no. 50-21-5, 85% purity) two weeks after thawing.
DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54. Only cells which passed the reactivity check were used for the assay.

-Plate Preparation
THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 106 cells/mL for 48 hours or at a density of 0.1 x 106 cells/ml for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 96 well flat-bottomed plate (80 µL/1.6 x 105 cells per well) for the dose finding assay or into a 24-well plate (500 µL/1 x 106 cells per well) for the expression measurements.

-Study Design
-Dose Finding Assay
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24 hours at 37°C, 5% CO2.
After the 24-hour incubation period, all cells from a 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL).
PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
Cell viability was calculated using the following equation:
Cell Viability =(number of living cells/total number of acquired cells)x100
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
Log CV75 = [(75 - c) x Log (b) - (75-a) x Log (d)]/ (a - c)
Where:
a was the minimum value of cell viability over 75% in testing groups
c was the maximum value of cell viability below 75% in testing groups
b and d were the concentrations showing the value of cell viability a and c respectively.

-CD86/CD54 Expression Measurement
One experiment (consisting of two independent runs) was needed to drive a prediction. Each independent run was performed on a different days.
On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 106 cells per well).
The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 hours at 37°C, 5% CO2.
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4ºC for 15 minutes.
After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes).
After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4ºC for 30 minutes.
The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).
The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

-Test Article
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 CAS Number Storage Batch Number Retest Date Purity
Dermol TDSA 19666-16-1 15 to 25°C, protected from light P7202 October 2019 100%

A certificate of analysis for the test article was provided by the Sponsor, see Certificate of Analysis.
The solvent control was isopropanol.
The positive control was 2,4-dinitrochlorobenzene supplied by Sigma-Aldrich Chemical Co. Ltd.

-Test Article Formulation
-Dose Finding Assay
The test article was found to be insoluble in dimethyl sulfoxide and saline, but was found to be soluble in isopropanol.
This vehicle produced a visually clear solution at a concentration of 500 mg/mL. Isopropanol is a known non-sensitiser, has been used in-house in human Cell Line Activation Tests with no impact on the performance or reliability of results and was therefore considered to be an appropriate solvent for this assay type.
The test article was dissolved at 500 mg/mL in isopropanol then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent.
The stock solutions were then further diluted 250-fold in culture medium (working solutions).
During the dilution process, an oily emulsion formed in the lowest five concentrations whereas oily droplets formed on the surface of the medium at concentrations of 250, 500 and 1000 µg/mL. Each of the working solutions was mixed five times to aid solution prior to treatment.

-CD86/CD54 Expression Measurement
Based on the solubility issues seen if the dose finding assay, the test article was dissolved at 125 mg/mL in isopropanol then eight stock solutions were prepared by 1.2-fold serial dilutions using the corresponding solvent to give eight doses ranging from 34.89 to 125 mg/mL.
The stock solutions were then further diluted 250-fold into the culture medium (working solutions).
This gave a maximum working solution concentration for the CD86/CD54 expression measurements of 250 µg/mL.

-Data Evaluation
-Analysis of Results
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test article-treated cells were calculated according to the following equation:
RFI = [(MFI of test article-treated cells – MFI of test article-treated isotype control cells)/(MFI of solvent-treated cells – MFI of solvent-treated isotype control cells)]x100

The cell viability from the isotype control cells (stained with mouse IgG1 antibodies) was calculated using the following equation:
Cell Viability =(number of living cells/total number of acquired cells)x100

-Prediction Model
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h CLAT prediction is considered NEGATIVE:
• The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
• The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%).

-Calculation of Effective Concentration (EC) Values
For test articles predicted as POSITIVE, two EC values, the EC150 for CD86 and the EC200 for CD54, are calculated as follows:
EC150 (for CD86) = Bconcentration + [(150 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)]
EC200 (for CD54) = Bconcentration + [(200 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)].
Where:
Aconcentration is the lowest concentration in µg/mL with RFI >150 (CD86) or 200 (CD54)
Bconcentration is the highest concentration in µg/mL with RFI <150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI >150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI <150 (CD86) or 200 (CD54).

-Assay Acceptance Criteria
• The cell viabilities of medium and solvent control should be higher than 90%
• In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%)
• For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%
• For the test article, the cell viability should be more than 50% in at least four tested doses in each run.

-Negative Results
Negative results are acceptable only for test articles exhibiting a cell viability of less than 90% at 1.2 x CV75 (highest concentration). If the cell viability at 1.2 x CV75 is equal to or greater than 90% the negative results should be discarded and the dose selection should be refined by repeating the CV75 determination.
When 5000 µg/mL in saline (or medium), 1000 µg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of the test article, a negative result is acceptable even if the cell viability is above 90%.

Positive control results:

In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Cell viability:

no effect on viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No CV75 value was calculated, as there was no effect on viability.

Group:

test chemical

Run / experiment:

mean

Parameter:

other: relative fluorescence intensity (RFI)

Value:

110 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Other effects / acceptance of results:

All assay acceptance criteria were met.
The cell viabilities of medium and solvent control were higher than 90% in each independent run.
In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%). The assay was considered valid as this did not impact on the results obtained with the test article.
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all each independent runs. Cell viability was >50% in each independent run.
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

The relative fluorescence intensity (RFI) values for the test article were calculated as follows:

Concentration (µg/mL)	RFI (CD86)		RFI (CD54)
	Exp 1	Exp 2	Exp 1	Exp 2
69.77	123	102	124	122
83.72	108	91	131	110
100.47	120	95	118	128
120.56	110	97	112	115
144.68	119	98	110	119
173.61	105	97	119	120
208.33	102	89	118	117
250.00	111	88	106	117
Solvent control	105	105	91	100
Positive control	195	406	549	700

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, Dermol TDSA, was considered to be negative in the human Cell Line Activation Test.

Executive summary:

The study was conducted to investigate the potential of the test article to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). 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 isopropanol and a dose finding assay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75).No CV75 value was calculated as there was no effect on viability at the maximum attainable concentration (250 µg/mL).

Eight stock solutions were prepared by 1.2-fold serial dilutions using isopropanol to give eight doses ranging from 34.89 to 125 mg/mL. These stock solutions were then diluted 250-fold into the culture medium (working solutions).

Aliquots of 500 µL of each of the working solutionswere mixed 1:1 (v/v) with cell suspensions at 1 x 10⁶cells per well. Each plate was sealed using a plate sealer and then incubated at37±1°C, 5% CO₂in air, in a humidified environmentfor 24±0.5 hours.

After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4°C for 15 minutes.

After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate, centrifuged and then stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 minutes.

The stained cells were washed with FACS buffer and re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

The test article, was considered to be negative in the human Cell Line Activation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Sensitisation testing was conducted in vitro/in chemico, with a raft of different recognised tests examining different sensitisation mechanisms. All the three studies returned negative results . It was thus considered that the registered substance did not meet the criteria for classification as a skin sensitiser of the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available