DODECANEDIOIC ACID, 1,12-BIS(2-OCTYLDODECYL) ESTER

Endpoint summary

Description of key information

Based upon results  Dioctyl dodecyl dodecanedioate is predicted as a non­ sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11/03/2019 to 05/04/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Principles of method if other than guideline:

The Test item was dissolved in non-standard vehicle Acetone (0.1 %in-well concentration)

GLP compliance:

not specified

Remarks:

Under a GLP-like quality system performed, where instruments and equipment are calibrated and maintained according to the supplier, a strict protocol is followed, QC criteria are met and notes and raw data is archived in paper or electronic.

Type of study:

other: GARDskin

Details of test system:

other: GARDskin

Details on the study design:

STANDARD GARD ASSAY PROTOCOL
Cell line maintenance and seeding of cells for stimulation
The human myeloid leukemia-derived cell line SenzaCell (available through ATCC), acting as an in vitro model of human Dendritic Cell (DC), is maintained in a-MEM (Thermo Scientific Hyclone, Logan, UT) supplemented with 20% (volume/volume) fetal calf serum (Life Technologies, Carlsbad, CA) and 40 ng/ml recombinant human Granulocyte Macrophage Colony Stimulating Factor (rhGM-CSF) (Miltenyi Biotec, Germany). A media change during expansion is performed every 3-4 days. Working stocks of cultures are grown for a maximum of 16 passages or two months after thawing. For chemical stimulation of cells, exposed cells are incubated for 24 hat 37°C, 5% CO2 and 95% humidity.

Test items handling and assessment of cytotoxicity
All Test items were stored according to instructions from the Sponsor, to ensure stability of Test items. Test items were dissolved in DMSO or water, based on physical properties. As many Test Items will have a toxic effect on the cells, cytotoxic effects of Test Items were monitored. When Test Items were poorly dissolved in cell medium the maximum soluble concentration was assessed. The assayed Test Items were titrated to concentrations ranging from 1 µM to the maximum soluble concentration in cell media. For freely soluble Test Iterns, 500 µM was set as the upper limit of the titration range. For Test Items dissolved in DMSO, the in-well concentration of DMSO was 0.1%. After incubation for 24 hat 37°C, 5% CO2 and 95% humidity, harvested cells were stained with the viability marker Propidium Iodide (PI) (BD Bioscience, USA) and analyzed by flow cytometry. PI-negative cells were defined as viable, and the relative viability of cells stimulated with each concentration in the titration range was calculated as


Relative viability = fraction of viable stimulated cells / fraction of viable unstimulated cells * 100

For toxic Test Items, the concentration yielding 90% relative viability (Rv90) was used for the GARD Assay, the reason being that this concentration demonstrates bioavailability of the test substance used for stimulation, while not impairing immunological responses. For non-toxic Test Items, a concentration of 500 µM was used if possible. For non-toxic Test Items that were insoluble at 500 µM in cell media, the highest soluble concentration was used. Whichever of these three criteria was met, only one concentration will be used for gene expression analysis. The concentration to be used for any given chemical was termed the 'GARD input concentration'.

GARD Main Stimulation
Once the GARD input concentration for the Test Items was established, the cells were stimulated again, as described above, using the GARD input concentration only. In addition to the assayed Test Items a set of positive and negativecontrols were performed as reference and quality controls. The Test Items and controls were assayed in biological replicates (n 2), performed at different time-points and using different cell cultures. After incubation for 24 hat 37°C, 5% CO2and 95% humidity, cell culture was lysed in TRizol reagent (Life Technologies) and stored at -20°C until RNA was extracted. In parallel, stimulated cells were PI stained and analysed by flow cytometry to verify the expected relative viability.

Isolation of RNA
RNA isolation from lysed cells was performed using commercially available kits (Direct-Zol RNA MiniPrep, Zymo Research, Irvine, CA). Total RNA was quantified and quality controlled using BioAnalyzer equipment (Agilent, Santa Clara, CA).

GARDskin Endpoint Measurement
Gene expression analysis using Nanostring nCounter System
A total of 100 ng of RNA was used as sample input in a hybridization assay with the GPS (GARD Prediction Signature) specific Reporter CodeSet (Nanostring Technologies, Seattle, WA). The hybridized sample was prepared on chip using nCounter Prep Station and individual transcripts of the GPS were quantified using Nanostring Digital Analyzer (Nanostring Technologies, Seattle, WA).

Nanostring nCounter data acquisition and normalization
Raw data was exported from the Digital Analyzer and RCC-files were imported into the R environment for statistical computing (www.r-project.org). Raw data was normalized using a single-chip normalization algorithm based on gene specific Counts Per Total Counts (CPTC) algorithm.

Prediction model and data analysis
For assessment of skin sensitization, a Support Vector Machine (SVM) was modelled on a training data set corresponding to samples used for assay development. For a comprehensive overview of the training data set and methods, see Forreryd et al, 2016 (for further details on biomarker discovery, see Johansson et al., 2011). Batch variations between the training data set and the test data set were eliminated using the Batch Adjustment by Reference Alignment (BARA) method (Gradin et al., 2019), using unstimulated cells as a reference control. Each sample in the test set were assigned a Decision Value (DV), based on its transcriptional levels of the GPS biomarker signature. Any test substance with a mean DV o (n 2) isclassified as a skin sensitizer.



The Test item was stored and handled according to instructions provided by the Sponsor.

For Dioctyl dodecyl dodecanedioate deviations from standard GARD assay protocol was made, where the Test item was dissolved in non-standard vehicle Acetone (0.1 %in-well concentration).

TheTest item was assessed for solubility and cytotoxic effect in order to establish the GARD input concentration (concentration inducing 90% relative viability). The Test item showed neither solubility issues nor cell cytotoxicity, therefore the GARD input concentration was determined to 500 µM.

For the GARD prediction, all replicates of the Test items were assigned Decision Values, where a Positive and a Negative Decision Value predicts the Test item as a sensitizer and a non-sensitizer, respectively. To eliminate cell batch variations between the training data set and the test data, unstimulated cells (m:2) were included as reference controls as described in the Standard GARD Assay Protocol section in this report. TheGARD assay passes Quality Control when the positive and the negative controls are predicted as a Sensitizer (+) and a Non-sensitizer(-), respectively.

Vehicle / solvent control:

other: Acetone

Negative control:

other: DMSO

Positive control:

other: p-phenulendiamine (PPD)

Positive control results:

Positive control of p-phenulendiamine (PPD) showed decision value (mean ±SD) of 6.59 ± 0.39

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Decision Value

Value:

-1.01

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

The objective of this study was to evaluate Dioctyl dodecyl dodecanedioate provided by the Sponsor using the GARDskin assay for a binary prediction as a sensitizer or a non­ sensitizer.

Based upon results of this study, Dioctyl dodecyl dodecanedioate is predicted as a non­ sensitizer. However, since no cytotoxic effect is seen, the bioavailability of the Test item is uncertain. Therefore, the prediction of Dioctyl dodecyl dodecanedioate as a non-sensitizer should be interpreted with caution.

Results from the cell cytotoxicity screen and the obtained GARD input concentrations for the provided Test item is presented in Table 1.

Table 1. Test item details

Test item	Vehicle•	Max. screenII (µM)	Rv9om (µM)	GARD input concIV (µM)
Dioctyl dodecyl dodeca.n. edioate	Acetone	500	No cytotox	500

1. Deviation from standard GARD assay protocol using non-standard vehicle


2. The highest soluble concentration used in screening


3. Concentration of Test item inducing 90% Relative Viability


4. Concentration based on screen and Rv90

 

The non-standard vehicle (Acetone) and the Test item were predicted as GARDskin non-sensitizers.

 

Test item	Decision Value (Mean:tSD)	GARDskin Prediction	GARDskin assay Quality Control
Dioctyl dodecyl dodecanedioate	-1.01 ±0.72	Non-sensitizer	PASSED
Acetone	-0.78 ±0.82	Non-sensitizer	PASSED
Positive ctrl, PPD	6.59 ±0.39	Sensitizer	PASSED
Negative ctrl, DMSO	-1.51 ±0.30	Non-sensitizer	PASSED

Interpretation of results:

GHS criteria not met

Conclusions:

The objective of this study was to evaluate Dioctyl dodecyl dodecanedioate provided by the Sponsor using the GARDskin assay for a binary prediction as a sensitizer or a non­ sensitizer.

Based upon results of this study, Dioctyl dodecyl dodecanedioate is predicted as a non­ sensitizer.

Executive summary:

The objective of this study was to evaluate Dioctyl dodecyl dodecanedioate provided by the Sponsor to assess the sensitizing hazard using the GARDskin assay for binary prediction as a sensitizer or a non-sensitizer.

Based upon the results of this study, Dioctyl dodecyl dodecanedioate is predicted as a non­ sensitizer. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

No cytotoxic effects were seen