Isodecyl 3,5,5-trimethylhexanoate

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:

03 May 2017 - 13 November 2017

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:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Regulation 1223/2009, Article 18, restricts the use of in vivo studies on cosmetic raw materials (the sole use of this substance), therefore a recognised in chemico test was conducted on the test material.

Specific details on test material used for the study:

The test article, a clear colourless liquid, was identified as Isodecyl 3,5,5-trimethylhexanoate and was received at Covance as follows:
- Test Article - Isodecyl 3,5,5-trimethylhexanoate
- Storage - 15 to 25°C, protected from light
- Purity - UVCB 100%

Details on the study design:

- Objectives:
The study was conducted to quantify the reactivity of Isodecyl 3,5,5-trimethylhexanoate 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 all test article and co-elution samples contained small particles, therefore all samples were centrifuged at 400 g for 5 minutes.

- 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: 7 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

TEST ARTICLE, POSITIVE CONTROL ARTICLE AND PEPTIDES
The test article, a clear colourless liquid, was identified as Isodecyl 3,5,5-trimethylhexanoate (also known as Isodecyl Isononanoate/Wickenol) and was received at Covance as follows:
Test Article Storage Batch Purity
Isodecyl 3,5,5-trimethylhexanoate 15 to 25°C, protected from light UVCB - 100%

Cinnamic aldehyde (CAS No. 104-55-2, batch number MKBT8955V purity 99.1%, expiry 29 February 2020) was used as the positive control.
The peptides, cysteine (lot number P161108-LC180433 purity 95.92%) and lysine (lot number P160825-LC107617, purity 99.26%) were obtained from RS Synthesis, Louisville, Kentucky, USA.

- 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.
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.

Positive control results:

Cinnamic aldehyde (CAS No. 104-55-2, batch number MKBT8955V purity 99.1%, expiry 29 February 2020) was used as the positive control.
The mean percentage peptide depletion (PPD) values for the positive control were:
- 55.93 for lysine
- 70.73 for cysteine

Run / experiment:

other: 1-3

Parameter:

other: % peptide depletion (PPD) - lysine

Value:

-3.42

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: The Max PPD value across 3 runs was 3.65, with a mean PPD of 1.22 for the 3 runs

Run / experiment:

other: 1-3

Parameter:

other: % peptide depletion (PPD) - Cysteine

Value:

1.89

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: he Max PPD value across 3 runs was 7.51, with a mean PPD of 5.16 for the 3 runs

Other effects / acceptance of results:

Lysine Depletion
The percentage peptide depletion values are shown in Table 6.1
The r2 value for the standard calibration curve was 0.9998.
The peptide concentrations for the Reference Controls A and C are shown in Table 6.2
The peak area results for Reference Controls B and C are shown in Table 6.3

Cysteine Depletion
The percentage peptide depletion values are shown in Table 6.4
The r2 value for the standard calibration curve was 0.9943.
The peptide concentrations for the Reference Controls A and C are shown in Table 6.5
The peak area results for Reference Controls B and C are shown in Table 6.6

Table 6.1 - PPD (Lysine)

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	34.356	35.658	3.65	1.22	2.1
	36.782		-3.15
	36.879		-3.42
Positive Control	15.189	35.658	57.4	55.9	3.3
	14.888		58.25
	17.066		52.14

Table 6.2 - Peptide concentrations for the Reference Controls A & C (Lysine)

Reference Control	Peptide Concentration (mM)			Mean
	Replicate 1	Replicate 2	Replicate 3
A	0.487	0.489	0.483	0.49
C	0.484	0.494	0.489	0.49

Table 6.3 - Peak area results for Reference Controls B & C (Lysine)

Reference Control	Replicate	Peptide Peak Area
B	1	38.123
	2	36.035
	3	36.385
	4	35.96
	5	36.133
	6	36.032
C	1	35.306
	2	36.018
	3	35.649
Mean		36.182
SD		0.79
CV		2.18

Table 6.4 - PPD (Cysteine)

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD
Test Article	25.009	25.49	1.89	5.16	2.9
	23.937		6.09
	23.575		7.51
Positive Control	7.667	25.49	69.92	70.7	0.7
	7.411		70.93
	7.303		71.35

Table 6.5 - Peptide concentrations for the Reference Controls A & C (Cysteine)

Reference Control	Peptide Concentration (mM)			Mean
	Replicate 1	Replicate 2	Replicate 3
A	0.546	0.545	0.548	0.55
C	0.523	0.526	0.496	0.51

Table 6.6 - Peak area results for Reference Controls B & C (Cysteine)

Reference Control	Replicate	Peptide Peak Area
B	1	26.879
	2	26.892
	3	26.6
	4	24.656
	5	24.965
	6	22.758
C	1	25.894
	2	26.058
	3	24.518
Mean		25.479
SD		1.38
CV		5.42

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, Isodecyl 3,5,5-trimethylhexanoate, was considered to be negative in the Direct Peptide Reactivity Assay.

Executive summary:

The study was conducted to quantify the reactivity of Isodecyl 3,5,5- trimethylhexanoate 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 nonsensitisers 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 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 5.16%, the lysine depletion value was 1.22% and the mean of the cysteine and lysine depletion values was 3.19%.

The test article, Isodecyl 3,5,5-trimethylhexanoate, was considered to be negative in the Direct Peptide Reactivity Assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 April 2017 - 25 October 2017

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:

ARE-Nrf2 luciferase KeratinoSens™ test method

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 colourless liquid, was identified as Isodecyl 3,5,5-trimethylhexanoate and was received at Covance as follows:
- Test Article - Isodecyl 3,5,5-trimethylhexanoate
- CAS Number - 59231-35-5
- Storage - 15 to 25°C
- Purity - 100%

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

- Objectives:
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.
- Test Article Formulation:
Test formulations were prepared using dimethylformamide (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, 0.195, 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.

- 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.

Vehicle / solvent control:

DMSO

Positive control results:

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.

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

8.87 µM

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

The EC1.5 values for the positive control was 14.14 μM in Experiment 3.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

16.98 µM

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

The EC1.5 value for the positive control was 25.57 μM

Remarks on result:

other:

Remarks:

The maximal fold increase (Imax) for Experiment 1 was 3.79

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

0.98 µM

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks:

The EC1.5 value for the positive control was 34.40 μM in Experiment 2. Experiment 2 did not therefore meet the acceptance criteria specified in the protocol.

Remarks on result:

other:

Remarks:

The maximal fold increases (Imax) was 3.06 Experiment 2.

Other effects / acceptance of results:

- Calculation of Imax and EC1.5 Values:
Luminescence readings and fold increases are given in Table 10.1, Table 10.2 and Table 10.3.
The maximal fold increases (Imax) were 3.79, 3.06 and 2.90 for Experiments 1, 2 and 3, respectively.
The EC1.5 values were 16.98, 0.98 and 8.87 μM for Experiments 1, 2 and 3, respectively.

- Viability:
MTT-absorbance readings are given in Table 10.4.
Cell viability at the lowest concentration with induction of luciferase activity above 1.5 fold was 94.7%, 43.9% and 103.4% for Experiments 1, 2 and 3, respectively.The IC50 and IC30 values could not be calculated as for Experiments 1 and 3 as the minimum viability did not drop below 70%.
The IC50 and IC30 values could not be calculated as for Experiment 2 as all viability
results were below 50%.

- Assay Acceptance:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 32 and 64 μM in Experiment 1, at a concentration of 64 μM in Experiment 2 and at concentrations of 16 to 64 μM in Experiment 3.
The EC1.5 values for the positive control were 25.57, 34.40 and 14.14 μM in Experiments 1, 2 and 3, respectively. Experiment 2 did not therefore meet the acceptance criteria specified in the protocol.
The average induction in the three replicates for the positive control at 64 μM was 2.65, 2.72 and 3.93 in Experiments 1, 2 and 3, respectively.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 8.37%, 8.75% and 9.01% in Experiments 1, 2 and 3, respectively.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In each experiment, the Imax was >1.5, EC1.5 value was <1000 μM and there was an apparent overall dose response for luciferase induction. In Experiments 1 and 3, cell viability at the lowest concentration with induction of luciferase activity above 1.5-fold was >70%.
As all 4 of the conditions were met in two out of three experiments, the test article, Isodecyl 3,5,5-trimethylhexanoate, was considered to be positive in the ARE-Nrf2 Luciferase Test.

Executive summary:

The study was conducted to investigate the potential of Isodecyl 3,5,5 -trimethylhexanoate 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 concentration (200 mM). Serial dilutions were then made using DMF to obtain 12 master concentrations of the test article (0.098, 0.195, 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) CO2 in air, in a humidified environment for 48±1 hours.

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

After the 48-hour exposure period, the cells in the viability plate 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 maximal fold increases (Imax) were 3.79, 3.06 and 2.90 for Experiments 1, 2 and 3, respectively.

The EC1.5 values were 16.98, 0.98 and 8.87 μM for Experiments 1, 2 and 3, respectively.

Cell viability at the lowest concentration with induction of luciferase activity above 1.5 fold was 94.7%, 43.9% and 103.4% for Experiments 1, 2 and 3, respectively. The IC50 and IC30 values could not be calculated as for Experiments 1 and 3 as the minimum viability did not drop below 70%. The IC50 and IC30 values could not be calculated as for Experiment 2 as all viability results were below 50%.

In each experiment, the Imax was >1.5, EC1.5 value was <1000 μM and there was an apparent overall dose response for luciferase induction. In Experiments 1 and 3, cell viability at the lowest concentration with induction of luciferase activity above 1.5-fold was >70%.

As all 4 of the conditions were met in two out of three experiments, the test article, Isodecyl 3,5,5-trimethylhexanoate, was considered to be positive in the ARE-Nrf2 Luciferase Test.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2017 - 06 November 2017

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 colourless liquid, was identified as Isodecyl 3,5,5-trimethylhexanoate and was received at Covance as follows:
- Test Article - Isodecyl 3,5,5-trimethylhexanoate
- Storage - 15 to 25°C, protected from light
- Purity - UVCB 100%

Details on the study design:

-Specifications:
Human monocytic leukemia cell line, THP-1 (an immortalised human monocyticleukemia 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 2,4-dinitrochlorobenzene (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 24-well flat-bottomed plate (80 µL/1.6 x 105 cells per well).

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 x 100
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:
The prediction was derived from three valid, independent runs. Each independent run was performed on a different day. On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL.Thecellswere 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 Formulation
- Dose Finding Assay:
The test article was dissolved at 100 mg/mL in DMF then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent.
The stock solutions were then further diluted 50-fold in culture medium (working solutions).

- CD86/CD54 Expression Measurement:
The test article was dissolved at 100 mg/mL in DMF then eight stock solutions were prepared by 1.2-fold serial dilutions using the corresponding solvent.
The stock solutions were then further diluted 250-fold into the culture medium (working solutions).

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.

Positive control results:

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54 in all 3 independent runs. Cell viability was >50% in each independent run.

Run / experiment:

other: Exp 1-3

Parameter:

other: relative fluorescence intensity

Remarks:

Surface Marker CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: The RFI for CD86 was <150% in 2 of 3 independent runs

Run / experiment:

other: Exp 1-3

Parameter:

other: relative fluorescence intensity

Remarks:

Surface marker CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: The RFI for CD54 was <200% in all 3 independent runs

Run / experiment:

other: Exp 1-3

Parameter:

other: Cell viability

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: Cell viability was >90% in all 3 independant runs

Other effects / acceptance of results:

- Dose Finding Assay:
The cell viability results are given in Table 7.1. It should be noted that the data were obtained from repeat experiments, as the original data were invalidated.
No toxicity was noted at the maximum concentration, therefore the CV75 value for the test article was >1000 μg/mL.

- CD86/CD54 Expression Results:
Geometric mean fluorescence intensity (MFI) and viability results are given in Table 7.2. It should be noted that the data for Experiments 1 and 2 were obtained from repeat experiments, as the original data were invalidated.
The RFI for CD86 was <150% in 2 of 3 independent runs.
The RFI for CD54 was <200% in all 3 independent runs.
Cell viability was >90% in all 3 independant runs.

- Assay Acceptance Criteria Results:
The cell viabilities of medium and solvent control were higher than 90% in all 3 independant runs.
In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) except in Experiment 3 where the RFI for CD86 was 168%. 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 3 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 3	Exp 1	Exp 2	Exp 3
279	93	149	90	87	97	91
334.8	88	159	81	91	95	83
401.8	68	158	83	86	92	76
482.2	83	218	81	86	93	86
578.7	77	184	68	78	90	64
694.4	74	180	76	83	86	87
833.3	78	166	76	77	80	85
1000	70	186	63	77	76	84
Positive Control	317	167	389	484	168	1594

RFI                                        Relative Fluorescence Intensity

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, Isodecyl 3,5,5-trimethylhexanoate, was considered to be negative in the human Cell Line Activation Test under the conditions of this study.

Executive summary:

The study was conducted to investigate the potential ofIsodecyl 3,5,5-trimethylhexanoateto 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 dimethylformamide (DMF) and a dose finding asssay was conducted to determine a concentration showing 75% THP-1 cell survival (CV75).

No toxicity was noted in the dose finding assay at the maximum concentration, therefore the CV75 value for the test article was >1000 µg/mL.Eight stock solutions were prepared by 1.2-fold serial dilutions using DMF. 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 with cell suspensions at 1 x 10⁶cells per well. 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 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 (or sample tubes), 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.

No toxicity was noted in the dose finding assay at the maximum concentration, therefore the CV75 value for the test article was >1000 µg/mL.

In the main study, the RFI for CD86 was <150% in 2 of 3 independent runs, the RFI for CD54 was <200% in all 3 independent runs and cell viability was >90% in all 3 independant runs.

All assay acceptance criteria were met except in Experiment 3 where the RFI for CD86 was 168% for the solvent control. The assay was considered valid as this did not impact on the results ontained with the test article.

The test article,Isodecyl 3,5,5-trimethylhexanoate, was considered to be negativein the human Cell Line Activation Test under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Sensitisation testing was conducted in vitro/in chemico, with a raft of different recognised tests examining different sensitisation mechanisms. Two of the three studies returned negative results (direct peptide binding and dendric cell activation) while the third (ARE-Nrf2 activation) provided a positive result. As only one of the studies presented recorded a positive result, it was 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).