Resin acids and Rosin acids, esters with glycerol and diethylene glycol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

Data Analysis Section 7.5.6 of the protocol describes the steps followed in typical data analysis. They include a description of the gates applied to the data to determine viability and geometric mean. The test article, MTDID 60827, however required an addition gating step which is not described in this section. The additional gating was required to obtain more accurate data due to observed precipitate in the dot plots in the Flow Cytometer. Without the additional gate, the analysis applied to cells and test article residue was skewed by the values collected for the residual test article. The h-CLAT is designed to measure the presence of CD86 and CD54 on the surface of the cells; therefore, it is important to isolate the cell population when performing the data analysis. In the case of this test article, an additional gate was necessary to eliminate the very small particles of debris (presumably residualtest article) remaining in the well at the time of data collection. Therefore, there was a significant effect on the procedure performed and those additional procedural steps (gating) resulted in a significant change in the experimental results. These changes were necessary to ensure the data analyzed was relevant to cellular expression of CD54 and CD86. There was no significant effect on the overall quality, integrity, and outcome of the study.

Deviations:

yes

Remarks:

See 'Version/remarks' field for the description of the deviations.

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

The Human Cell Line Activation Test (h-CLAT) was used to assess the skin sensitization potential of the
test article by monitoring the upregulation of cell surface markers, CD54 and CD86, on the surface of
human acute monocytic leukemia cells (THP-1). The upregulation of CD54 and CD86 in response to a skin
sensitizer is correlated to dendritic cell activation, which is the third key event of the skin sensitization
pathway.

Test material

Test material information

Constituent 1

Reference substance name:

Resin acids and Rosin acids, esters with glycerol and diethylene glycol

EC Number:

288-327-7

EC Name:

Resin acids and Rosin acids, esters with glycerol and diethylene glycol

Cas Number:

85711-66-6

Molecular formula:

UVCB substance

IUPAC Name:

esterification product of (1R,4aR,4bR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylic acid; (1R,4aR,4bS,10aR)-1,4a-dimethyl-7-(propan-2-ylidene)-1,2,3,4,4a,4b,5,6,7,9,10,10a-dodecahydrophenanthrene-1-carboxylic acid; (1R,4aS,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,5,6,9,10,10a-decahydrophenanthrene-1-carboxylic acid; (1R,4aS,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylic acid and 2,2'-oxydiethanol and propane-1,2,3-triol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M, lot: K02007
- Purity, including information on contaminants, isomers, etc.: no data.


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 30 °C (Room Temp)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: no data
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The top stock dilution of the test article was found to be non-soluble at 100 mg/mL in Saline or at 500 mg/mL in DMSO. Therefore, the test article final stock was attempted to be solubilized at a concentration at 10 mg/mL directly in THP-1 Culture Medium
- Reactivity of the test material with the incubation material used (e.g. plastic ware): no data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Grinding into poweder, vortexing and sonicating.
- Preliminary purification step (if any):
- Final concentration of a dissolved solid, stock liquid or gel: 39 to 5000 µg/mL
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): no data.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: The test article stock and working dilutions were observed to be cloudy pink with white particles non-viscous suspension.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added:

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

442E

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution
: test article will be dissolved to the maximum appropriate concentration determined in the solubility test, or up to a maximum contration of 100 mg/ml in saline, or a maximum concentration of 500 mg/ml in DMSO.
- Preparation of the test chemical serial dilutions
: seven serial doses using a typical dilution factor of 1.2 to 1.5 will be prepared such that 8 doses will be tested in the definitive assay.
- Preparation of the positive controls
: 2,4-Dinitrochlorobenzene at stock concentration of 2 mg/ml
- Preparation of the solvent, vehicle and negative controls : the solvent control will be culture medium for test articles diluted in saline or culture medium. The solvent control will be DMSO in culture medium for test articles diluted in DMSO. A single concentration of the solvent control(s) will be prepared in culture medium and dosed on the cells in the same manner as the test article(s) so the final concentration of saline on the cells is 1% and DMSO is 0.2%.
- Stable dispersion obtained

DOSE RANGE FINDING ASSAY:
- Highest concentration used
: 500 mg/ml
- Solubility in solvents
: The top stock dilution of the test article was found to be non-soluble at 100 mg/mL in
Saline or at 500 mg/mL in DMSO. Therefore, the test article final stock was attempted to be solubilized
at a concentration at 10 mg/mL directly in THP-1 Culture Medium, after grinding the test article into a
powder, and then vortexing and sonicating. Using this strategy, the final concentration of the test article
on the cells ranged from 39 to 5000 µg/mL.
- Cytotoxicity assessment performed
A preliminary (dose range finding assay) was performed to determine the viability of the THP-1 cells after
24 ± 0.5 hour exposure to 8 test article concentrations. The CV75, which is the calculated test article
concentration leading to 75% cell viability resulted in 2230 µg/mL.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 8

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density)
: cells resuspended in fresh medium to a density of 2.0 x 10^6 cells/ml and 500 microliters of the cell supsension will be seeded into the appropriate wells. Resulting in 1.0x10^6 cells/well.
- Incubation conditions
: 37 degree in a humidified atmosphere of 5 (+/-) 1% CO2 in air.
- Washing conditions
: Centrifugation 200 to 300 g for 5 minutes at 4 degrees C.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used : MACSQuant Analyzer (Miltenyi) with three laser system capable of both FITC and PI acquisition
- Plate used : 24 well plate
- Propidium iodide staining/cytotoxicity measurements : After 24 hours of exposure and cell rinsing, Propidium Iodide was added to the samples to a final concentratino of 0.625 ug/mL.
- Preparation for CD54 and/or CD86 expression measurements/cell staining : PI uptake was analyzed via flow cytometry. Cells stained with PI represent the non-viable cell population and will be gated out to identify the viable populations.

Vehicle / solvent control:

cell culture medium

Negative control:

not applicable

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Value:

1 239 µg/mL

Cell viability:

Cell viability was approximately 93%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC200, CD54 [442E]

Value:

822 µg/mL

Cell viability:

Cell viability was approximately 94%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: CD 86 RFI > 150

Remarks:

CD 86 RFI > 150. The lowest concentration tested (754 ug/mL) elicited an RFI of 169.

Value:

754 µg/mL

Cell viability:

94.45%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The highest concentration (2250 ug/mL) elicted an RFI of 80.59 so an EC150 could not be calculated for this run.

Results 5

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Value:

2 230 µg/mL

Outcome of the prediction model:

positive [in vitro/in chemico]

Applicant's summary and conclusion

Interpretation of results:

other: See 'Remarks'

Remarks:

The test article is predicted to have skin sensitization potential within Key Event 3 (KE3) of the skin sensitization adverse outcome pathway (AOP).

Conclusions:

Based on the results of the study (EC200 = 822 ug/mL and EC150 <750 ug/mL), rosin acid derivative is predicted to have skin sensitization potential within Key Event 3 (KE3) of the skin sensitization adverse outcome pathway (AOP).

Executive summary:

 The skin sensitization potential of rosin acid derivatives was evaluated in the h-CLAT assay. The study was conducted according to OECD 422E in compliance with OECD GLP. The sensitization potential of rosin acid derivatives was evaluated by measuring the changes in the expression of cell surface markers CD54 and CD86 associated with the process of dendritic cell activation in the human leukemia cell line, THP-1, following exposure to a test article. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescently labelled antibodies for CD54 and CD86. The upregulation of CD54 and CD86 in response to a skin sensitizer is correlated to dendritic cell activation, which is the third key event of the skin sensitization pathway. The test article was solubilized directly in THP-1 culture medium, after grinding the test article into a powder, then vortexing and sonicating. The final concentration of the test article on the cells ranged from 39 to 5000 ug/mL. THP-1 cells were seeded at a density of 2.0 x 10^6 cells/mL in culture medium in 24 -well plate format. Cells were exposed to rosin acid derivative at 754, 904, 1085, 1302, 1563, 1875, 2250, and 2700 ug/mL, the positive control (2,4-Dinitrochlorobenzene at 2 mg/ml) or negative control (culture media). Following a 24-hour exposure, cytotoxicity was assessed via Propidium Iodine (PI) staining. Cells were incubated with CD54 and CD86 antibodies for 30 minutes. Following incubation, CD54 and CD86 expression as well as PI uptake (cytotoxicity measurement) was measured via flow cytrometry. The h-CLAT is designed to measure the presence of CD86 and CD54 on the surface of the cells; therefore, it is important to isolate the cell population when performing the data analysis. In the case of this test article, an additional gate was necessary to eliminate the very small particles of debris (presumably residual test article) remaining in the well at the time of data collection. Therefore, there was a significant effect on the procedure performed and those additional procedural steps (gating) resulted in a significant change in the experimental results. These changes were necessary to ensure the data analyzed was relevant to cellular expression of CD54 and CD86. There was no significant effect on the overall quality, integrity, and outcome of the study.

 

The results from the first assay performed were deemed invalid due to the positive control values did not meet validation criteria. Two definitive assays were performed following the first assay and only results from the two valid assays have been reported. Based on the results of the assays, rosin acid derivative had CD54 EC200 values of 1239 and 822 ug/mL and CD86 EC150 values of <750 and >1875 ug/mL in the first and second experiments, respectively. Based on the results of the study (EC200 = 822 ug/mL and EC150 <750 ug/mL), rosin acid derivative is predicted to have skin sensitization potential within Key Event 3 (KE3) of the skin sensitization adverse outcome pathway (AOP).