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

Endpoint summary

Description of key information

The Direct Peptide Reactivity Assay (DPRA), Keratinosens, and h-CLAT assays were conducted with rosin acid derivatives. The results of the studies were:

DPRA: Minimally reactive when tested according to OECD 442C

Keratinosens: Negative when tested according to OECD 442D

h-CLAT: Positive when tested according to OECD 442E.

Utilizing the 2 out of 3 Defined Approach (2o3 DA) outlined in OECD 497, rosin acid derivatives should not be classified for skin sensitization.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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.

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:

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]

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

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

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

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.

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]

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).

Reference 2

Endpoint:

skin sensitisation: in chemico

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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 Company, lot # K02007
- Purity, including information on contaminants, isomers, etc.:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: no data
- 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: no data
- 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): solid test article was ground and solubolized in 1:1 Acetonitrile:Aceton
- Preliminary purification step (if any): None
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): ground to fine powder

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solid test article was ground and solubolized in 1:1 Acetonitrile:Acetone

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
Solid test article was ground and solubolized in 1:1 Acetonitrile:Acetone to 40 mg/mL
- Preparation of the positive controls
: 100 mM cinnamic aldehyde prepared in acetonitrile
- Preparation of the solvent, vehicle and negative controls : no data
- Stable dispersion obtained
: no data
- Other: no data

DOSE RANGE FINDING ASSAY:
- Highest concentration used
: 40 mg/mL
- Solubility in solvents
: soluble in 1:1 Acetonitrile:Aceton
- Final concentration range selected on basis of: per guideline

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates
: three
- Number of repetitions: one
- Test chemical concentrations
: 40 mg/mL
- Application procedure
: not applicable
- Exposure time: not applicable
- Study evaluation and decision criteria used
: DPRA predication model as per OECD 442C (2020)
- Description on study acceptance criteria
:
1) Each standard curve must have an r2 > 0.990 and the mean peptide concentration of the
reference controls A and C must equal 0.50 ± 0.05 mM. The peak area CV for reference controls
B and C must be < 15%
Project 11288, Final Report Page 8 of 33
2) The percent peptide depletion for the cysteine peptide exposed to the positive control (cinnamic
aldehyde) must be > 60.8% and < 100.0% and the SD for the replicates must be < 14.9. For the
lysine peptide, the percent depletion by the positive control must be > 40.2% and < 69.0% and
the SD for the replicates must be < 11.6.
3) The SD criteria for the test substance replicates must be < 14.9% for the percent cysteine
depletion and <11.6% for the percent lysine depletion.


DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

1:1 mix, acetone:acetonitrile

Positive control:

cinnamic aldehyde

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

2 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.15 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the study, rosin acid derivatives are not predicted to have skin sensitization potential within the Key Event 1 (KE1) stage of the adverse outcome pathway (AOP).

Executive summary:

Rosin acid derivatives were evaluated for skin sensitization potential using the Direct Peptide Reactivity Assay (DPRA). The study was conducted according to OECD 442C in compliance with OECD GLP. The test article was solubilized in 1:1 Acetonitirile: Acetone with vortexing and evaluated in 3 definitive trials. Since the test article is a solid, it was ground with a mortar and pestle prior to performing the dilution from the final top concentration of 40 ug/mL. Due to deviations that occurred in the first and second trial, only the results from the third trial were considered valid and were evaluated. The cysteine peptide was prepared at 0.667 mM in cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer as outlined in OECD 442C. Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile containing no reference or test articles. After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. The mean peptide depletion (%) of cysteine and lysine was 1.57 and the mean cysteine depletion (%) was 2.00. Based on the results of the study, rosin acid derivatives are not predicted to have skin sensitization potential within the Key Event 1 (KE1) stage of the adverse outcome pathway (AOP)..

Reference 3

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

During the conduct of the first definitive trial initiated on 13 October 2020, the following three protocol deviations occurred.
Cells were inadvertently placed in the well designated as a blank. The cells were removed and
the well was washed twice before 1% DMEM was placed in the well.
The 96-well plate designated for the MTT viability endpoint was dropped immediately after the
test and control articles were dosed and the plate sealed with an adhesive plate seal. Since no
dosing solutions were observed to be lost, the plate was placed into the incubator and the assay
was continued.
After the completion of the 48±1 hour treatment incubation, the three plates designated for the
luciferase gene induction endpoint were allowed to equilibrate to room temperature for only 23
minutes prior to decanting the dosing solutions. This is a deviation from the protocol which states
that the plates will be allowed to cool to room temperature for at least 30 minutes.
After the completion of the 48±1 hour treatment incubation, the plate designated for the MTT
viability endpoint was removed from the incubator 30 minutes beyond the specified 48±1 hour
timeframe.
Due to these deviations, at the Study Director’s judgement, the results from the first definitive trial were
not considered reliable and additional trials were conducted. Although the control results fell within the criteria for a valid test, the test results were not included in the overall evaluation of sensitization
potential.

Deviations:

yes

Remarks:

See 'Version/remarks'

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

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 Company, 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), protected from exposure to light.
- 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: soluble in DMSO.
- 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): no data.
- Preliminary purification step (if any):
- Final concentration of a dissolved solid, stock liquid or gel: test article prepared at 100X and diluted to 4X. The 4X dilutions wll result in a 1X final dilution after direct addition of 50 µliters of each 4X test article to the cell cultures containing 150 µL
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle):

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: no data.


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:no data.

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical: The 4X stock solution was prepared to a top concentration of 1.6 mg/mL in 4% DMSO in 1% DMEM.
- Preparation of the positive controls : cinnamic aldehyde will be serially diluted in DMSO to concentrations 100x of the final concetrations for the assay. The final 1X concentrations of the positive control will be: 64, 32, 16,8, and 4 µM
- Preparation of the solvent, vehicle and negative controls : solvent control- each plate will include 6 solvent control wells (cells exposed to 1% DMSO only). If an intermediate solvent is used (e.g. water) the concentration of the intermediate solvent may be the same as used with the test article treatment.

DOSE RANGE FINDING ASSAY:
- Highest concentration used : 400 ug/mL
- Solubility in solvents : Not reported
- Solubility in incubation medium : No preciptiation reported.
- Cytotoxicity assessment performed : yes, cell viability was determined compared to solvent control using an MTT assay.
- Final concentration range selected on basis of: Maximum recommended concentration per OECD 442D.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates : Two definitive independent assays were conducted.
- Number of repetitions : tested in a total of five definitive assays (three trials initiated 13
October 2020, and two trials initiated 3 November 2020). Each definitive assay included a set of 4 plates
(3 for gene induction, 1 for cytotoxicity assessment). The third definitive trial was not valid since the
standard deviation of the luminescence readings for the solvent controls exceeded 20%.
- Test chemical concentrations : tested at 12 concentrations ranging from 0.195 to 400 µg/mL
- Application procedure
- Exposure time : 48 hours
- Study evaluation and decision criteria used : Per OECD 442D.
- Description on study acceptance criteria : Per OECD 442D.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density) : 1.0 x 10^5 cell/mL in assay medium. Cells are maintened for up to 25 passages after thawing.
- Incubation conditions : 37 C, 5% CO2
- Washing conditions : Rinsed twice with approximately 10 mL of DPBS containing EDTA for approximately 2 minutes.
- Precipitation noted : None noted in the study report.:

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometert: Molecular Devices SpectraMax luminometer
- Plate used : 96 well

DATA EVALUATION
- Cytotoxicity assessment : MTT direct reduction test
- Prediction model used : Per OECD 442D.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

245.75 µg/mL

Cell viability:

>70% (IC30 > 400 ug/mL)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Cell viability:

>70 % (IC30 > 400 ug/mL)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

A 1.5-fold increase in luciferase activity was not observed in this trial.

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

336.79 µg/mL

Cell viability:

> 70% (IC30 > 400 ug/mL)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

A test article will be considered to have sensitization potential if:
1) the EC1.5 value falls below 200 µg/mL in at least 2 of 3 repetitions,
2) at the lowest concentration with a gene induction above 1.5, cellular viability is > 70%,
3) there is an apparent overall dose response which is similar between repetitions.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results, rosin acid derivative is not predicted to have skin sensitization potential within Key Event 2 (KE2) of the skin sensitization adverse outcome pathway (AOP).

Executive summary:

The skin sensitization potential of rosin acid derivatives was evaluated in the Keratinosens assay. The study was conducted according to OECD 442D in compliance with OECD GLP. Rosin acid derivatives were dissolved in DMSO and each plate tested a range of 12 dosing concentrations ranging from 0.195 to 400 ug/mL. Positive (cinnamic aldehyde) and solvent (DMSO) controls were tested in parallel. Cells were exposed to the test article and controls for 48 hours after which cell viability (cytotoxicity) was determined via MTT assay and the luciferase activity was measured via incubation with ONEGlo Reagent and measurement of the subsequent luminescence via a Molecular Devices SpectraMax luminometer. Rosin acid derivatives were tested in a total of five definitive assays. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The third definitive trial was not valid since the luminescence reading for the solvent controls exceeded 20%. Only results from valid assays are reported. In the three definitive trials, rosin acid derivatives elicted EC1.5 values of 245.75 and 336.79 ug/mL. An EC1.5 value could not be calculated for one of the trials as 1.5 -fold induction of luciferase activity was not observed. Both EC1.5 values are > 200 ug/mL, both elicted greater than a 1.5 -fold increase in luciferase activity while also having cell viability above 70% (IC30 values of >400 ug/mL in all trials). Based on the Keratinosens decision tree published in OECD 497 (Annex 1, Figure 1.2), one trial would be negative, one borderline and one negative. Based on the results, rosin acid derivative is not predicted to have skin sensitization potential within Key Event 2 (KE2) of the skin sensitization adverse outcome pathway (AOP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Utilizing the 2 out of 3 Defined Approach (2o3 DA) outlined in OECD 497, rosin acid derivatives should not be classified for skin sensitization.