methyl 2-hydroxybut-3-enoate

Endpoint summary

Description of key information

The tests were conducted with the purified and stabilised (registered) form of the test substance. 

 

Using the Integrated Testing Strategy Defined Approach (ITSv2 DA) according to OECD guideline 497, the skin sensitising hazard potential as well as the potency-subcategorization according to UN GHS (subcategories 1A and 1B) of the substance was predicted. Therefore, three key events of the skin sensitisation AOP were tested experimentally and combined with the DASS Automated Workflow OECD QSAR Toolbox (v4.5) prediction.

 

First, three key events of the skin sensitisation AOP were tested experimentally. An in chemico skin sensitisation study according to OECD guideline 442C (DPRA, negative), an in vitro ARE-Nrf2 Luciferase Test according to OECD guideline 442D (KeratinoSens, positive) and an in vitro human Cell Line Activation Test according to OECD guideline 442E (h-CLAT, positive), in summary, predicted a skin sensitising potential of the test item. Using the DASS Automated Workflow in the OECD Toolbox, a skin sensitising potential was prediced.

 

Following the ITS data interpretation procedure, scores were assigned to the quantitative results from the DPRA and h-CLAT and to the OECD QSAR Toolbox v4.5 prediction (see table 1). 

 

The DPRA was valid and negative, thus, a score of 0 was assigned. In the h-CLAT, the increase of CD54 and CD86 marker expression was greater than 200% and 150% respectively in three valid independent experiments but the minimum induction threshold (MIT= min(EC150 CD86, EC200 CD54)) could not be calculated since no clear dose response could be observed. Thus, following a worst-case scenario, a maximum score of 3 was assigned to the h-CLAT results. Since the skin sensitisation QSAR prediction was positive and valid, it was assigned a score of 1. As two in vitro assays and the QSAR prediction were considered valid, the proposed categorisation according to OECD guideline 497 was used (table 2). In summary, a total battery score of 4 was calculated, predicting a sensitising pontential and potency of the substance allowing for classification into UN GHS Cat. 1B (moderate/weak sensitiser).

 

Table 1: Schematic of the ITS defined approach. Assignment of scores to test results.

Score	h-CLAT MIT (µg/mL)	DPRA Mean Cysteine and Lysine % depletion	DPRA Cysteine % depletion	In silico (ITSv1: DEREK; ITSv2: OECD TB)
3	≤10	≥42.47	≥98.24
2	>10, ≤150	≥22.62, <42.47	≥23.09, <98.24
1	>150, ≤5000	≥6.38, <22.62	≥13.89, <23.09	Positive
0	not calculated	<6.38	<13.89	Negative

 

Table 2: Prediction of sensitising potency based on total battery score

Potency	Total Battery Score
UN GHS 1A	6-7
UN GHS 1B	2-5
Not classified	0-1

 

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:

weight of evidence

Study period:

2021-06-16

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:

2019-06-18, corrected 2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
Cysteine peptide stock solution, 0.667 mM, 0.501 mg/mL: 0.01121 g of the peptide was pre-weighted and 20.158 mL of pH 7.5 phosphate buffer was added right before beginning the assay.
Lysine peptide stock solution, 0.667 mM, 0.518 mg/mL: 0.01033 g of the peptide was pre-weightedand 18.725 mL of pH 10.2 acetate buffer was added right before beginning of the assay.

- Preparation of the test chemical solutions: 100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.0234 g) based on the molecular weight and purity of the substance. 0.0234 g test chemical was weighted for the stock solutions used for the cysteine peptide depletion determination respectively and 0.0233 g test chemical was weighted for the stock solution used for lysine peptide depletion determination.

- Preparation of the positive controls, reference controls and co-elution controls
Positive control: 100 mM solutions of the positive control chemical in acetonitrile were prepared just before use. The needed amount of test chemical was calculated (0.0664 g ± 10%) based on the molecular weight and purity of the substance. 0.0663 g cinnamaldehyde was weighted for the positive stock solution used for the cysteine peptide depletion determination and 0.0661 g cinnamaldehyde was weighted for the stock solution used for lysine peptide depletion determination in the runs.
Reference control A: Peptide stock solutions are combined with acetonitrile. System suitability is checked by the use of the three replicates of reference control A.
Reference control B: Peptide stock solutions are combined with acetonitrile. Stability of the peptides are checked by the use of the three replicates of reference control B, measured before and after of the reaction samples.
Reference control C: Peptide stock solutions are combined with the respective solvent of the test item (and acetonitrile in case of cysteine peptide). Three replicates of reference control C are used as a solvent control to which the peptide concentration/depletion of the reaction samples is compared. Since acetonitrile is not the chosen solvent for the test item, a reference control C with acetonitrile is prepared additionally as the solvent control for the positive control.
Co-elution controls: Test item stock solution (and acetonitrile in case of cysteine peptide) is combined with the respective buffer solutions in each run. Co-elution controls are used to check for test item and peptide co-elution.

INCUBATION
- Incubation conditions: The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 22.5°C - 30°C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution
- Precipitation noted: No precipitation was noted.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Six calibration standard points were prepared by serial dilution of the peptide stock solutions with the following nominal molarities: STD 1 = 0.534 mM, STD 2 = 0.267 mM, STD 3 = 0.1335 mM, STD 4 = 0.0667 mM, STD 5 = 0.0334 mM and STD 6 = 0.0167 mM. As dilution buffer a 20% acetonitrile:buffer solution (phosphate or ammonium acetate) was used. For the zero standard point (STD 7 = 0 mM) dilution buffer was used.
- Verification of the suitability of the HPLC for test chemical and control substances: Prior to routine use of the method, the laboratory demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 200 nm

Vehicle / solvent:

water

Positive control:

cinnamic aldehyde

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 67.22 % ± 0.51 % and with lysine peptide depletion value of 52.07 % ± 1.33 %.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.88 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no relevant increase

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

1.24 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no relevant increase

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the method, the laboratory demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for reference controls A to C: Yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

study cannot be used for classification

Remarks:

The DPRA prediction must be considered in the framework of an IATA for a final prediction.

Conclusions:

In the in chemico Direct Peptide Reactivity Assay (DPRA) according to OECD guideline 442C, the test item showed no or minimal reactivity towards the synthetic peptides; thus it is not a potential skin sensitizer under the experimental conditions.

Executive summary:

In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA) in accordance with OECD 442C and GLP. For the test item and the positive control substance, in order to derive a prediction two independent tests were evaluated, one with cysteine and one with lysine peptides. The results of two valid runs is used for the classification of the test item. Peptide depletion by the positive control cinnamaldehyde was 67.22 % ± 0.51 % for cysteine peptides and 52.07 % ± 1.33 % for lysine peptides, fulfilling all acceptance criteria for the positive control.

 

The mean back-calculated peptide concentrations of the reference control A replicates were within the expected molarity concentration range for cysteine (0.49 mM) and lysine peptides (0.50 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.9 % and 0.2 % percentages for cysteine and lysine peptides, respectively. Moreover, the mean back-calculated peptide concentrations of the three reference controls C in the ultrapure water replicates were within the expected molarity concentration range for cysteine (0.47 mM) and lysine peptides (0.50 mM). For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 1.24 % ± 2.72 % while the percent lysine peptide depletion was 1.88 % ± 0.46 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 1.56 %, which is well below the threshold of 6.38 % for the applicable prediction model. The test item fells therefore into the no or minimal reactivity class and is evaluated not to be a skin sensitizer in this DPRA test.

 

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item showed no or minimal reactivity towards the synthetic peptides; thus it is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method according to OECD 442C.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-05-25 to 2021-07-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according 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)

Version / remarks:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: Saline was used to dissolve the test item for the stock solution. The test item was first diluted to the concentration corresponding to the CV75 × 1.2^3 value (43.1 μg/mL).
- Preparation of the test chemical serial dilutions: Ten concentrations were used in the main test. For the master solutions, 1.2-fold serial dilutions were made from the stock solution using saline (ten concentrations). The master solutions were then further diluted 50-fold into the culture medium to obtain the working solutions. These working solutions were finally used for exposure with a further final 2-fold dilution factor in the plate.
- Preparation of the positive controls: DNCB was used as the positive control at a final nominal concentration of 4 μg/mL on the plate. To obtain a 4 μg/mL concentration, a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to an 8 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells.
- Preparation of the solvent, vehicle and negative controls: Culture medium was used as negative control for the test item and to assess the impact of DMSO. Moreover, DMSO was used as a solvent control for the positive control (DNCB) at a single final concentration in the plate of 0.2%. To obtain a 0.2% concentration, DMSO was diluted 250-fold with culture medium to a 0.4% working solution. The working solution then was diluted 2-fold when added to the cells.
For the test item, the relative fluorescence intensity of surface markers was calculated compared to its relevant solvent/vehicle control.
- Stable dispersion obtained: Yes
- Log Kow of the test chemical: The extrapolated log Kow was determined to be 0.13.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 1000 μg/mL
- Solubility in solvents: Solubility of the test item was evaluated in saline at the concentration of 100 mg/mL. The test item could be properly dissolved in saline after 1 minute of vortexing and a clear, transparent and homogenous solution was obtained.
- Solubility in incubation medium: No precipitation was noted.
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75: A high level of cytotoxicity was observed in the first experiment in case of higher concentrations (1000 – 125 μg/mL). Thus, 43 μg/mL was used as highest final test item concentration on the plate and 1.2-fold dilutions were used in order to be able to determine the CV75 value more precisely in a second experiment. Since cytotoxicity was observed just at the highest tested concentration, a higher initial concentration (63 μg/mL) and 1.2-fold dilutions were used in the third experiment. In the third experiment, a biphasic dose response was seen. Here, CV75 values could be calculated either between 36.5 – 30.4 μg/mL or between 25.3 – 21.1 μg/mL. Due to this uncertainty, a fourth experiment was also performed where the same concentration range and
dilution was used as in the third one. The CV75 value could be calculated between 25.3 – 21.1 μg/mL. A similar biphasic dose response was noted as in the third experiment. Taken all data together, a CV75 value of 24.9 μg/mL was used for setting the dose range for measuring CD86 and CD54 expression in the main test.
- Final concentration range selected on basis of: A CV75 value of 24.9 μg/mL was used for setting the dose range for measuring CD86 and CD54 expression in the main test. However, due to the controversial findings concerning cytotoxicity during the preliminary test and the biphasic toxicity behavior, ten concentrations instead of eight doses was used in the main test. The final concentrations for the main test were 8.3, 10, 12, 14.4, 17.3, 20.8, 24.9, 29.9, 35.9 and 43.1 µg/mL.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 1
- Number of repetitions: 3
- Test chemical concentrations: 8.3, 10, 12, 14.4, 17.3, 20.8, 24.9, 29.9, 35.9 and 43.1 µg/mL
- Application procedure: Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 106 cells) at 1:1 ratio.
- Exposure time: 24±0.5 hours
- Study evaluation and decision criteria used: See "Any other information on materials and methods incl. tables"
- Description on study acceptance criteria: See "Any other information on materials and methods incl. tables"

SEEDING AND INCUBATION
- Seeding conditions: THP-1 cells were seeded at a density of either 0.1 × 10^6 cells/mL or 0.2 × 10^6 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintenance medium at 2 × 10^6 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 μL cell suspension / well (1 × 10^6 cells/well). The passage number was 10 at the start of the experiment and 14 at the end of the experiment.
- Incubation conditions: At 37°C under 5 % CO2
- Washing conditions: After incubation, cells were transferred from the 24-well plate into sample
tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by
centrifugation (300 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of
FACS buffer.

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
- Flow cytometry used: Beckman Coulter Flow Cytometer, CytoFLEX System B4-R0-V0
- Plate used: Sample tubes
- Propidium iodide staining/cytotoxicity measurements: After CD86 and CD54 staining, cells were washed twice with 150 μL of FACS buffer and were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. Cytotoxicity was checked based on the PI uptake and was analysed with the acquisition channel FL-3. A total of 10,000 living cells (PI negative) were acquired (when the cell viability was low, up to 30,000 cells including dead cells could be acquired). Alternatively, data could be acquired for one minute after the initiation of the analysis. The cell viability was automatically calculated by the cytometer analysis program.
- Preparation for CD54 and/or CD86 expression measurements/cell staining: Cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at approximately 4 °C for 30 min. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.

Vehicle / solvent control:

saline [442E]

Negative control:

other: culture medium

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The positive control gave expected results for both markers, with RFI values of both CD86
(537 %, 624 % and 391 % in the first, second and third experiment) and CD54 (422 %, 380 % and 385 % in the first, second and third experiment) expression higher than the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and with the respective cell viabilities being more than 50 % in each run (93.05 %, 94.58 % and 94.38 % in the first, second and third experiment).

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Value:

24.9 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC200, CD54 [442E]

Remarks:

The increase of CD54 marker expression (RFI) was greater than 200% compared to the respective negative controls in all three experiments. The EC200 was not determined, since no clear dose response could be observed.

Cell viability:

> 50 % of cell viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Remarks:

The increase of CD86 marker expression (RFI) was greater than 150 % compared to the respective negative controls in all three experiments. The EC150 was not determined, since no clear dose response could be observed.

Cell viability:

> 50 % of cell viability

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in Annex II of the Test Guideline 442E, the laboratory demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended and listed in APPENDIX II of the OECD TG 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges (Study Number: 392-442-5369). Moreover, a historical database of data generated with the reactivity checks and with the positive and solvent/vehicle controls is maintained over time to confirm the reproducibility of the test method in the laboratory.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

study cannot be used for classification

Remarks:

The h-CLAT prediction must be considered in the framework of an IATA for a final prediction.

Conclusions:

In a human Cell Line Activation Test (h-CLAT prediction model) according to OECD guieline 442E, the test item is concluded positive for skin sensitisation under the experimental conditions of this test.

Executive summary:

In the course of this in vitro human Cell Line Activation Test (h-CLAT) according to OECD 442E and GLP, the skin sensitisation potential of the test item was examined. The extent of cytotoxicity induced on THP-1 cells by the test item to allow a concentrationsetting for the main study was investigated in four dose finding tests. The maximal finalconcentrations used on the plates for the test item previously dissolved in saline were1000.0 μg/mL (first run), 43.0 μg/mL (second run), 63.0 μg/mL (third run) and 63.0 μg/mL (fourth run). Based on the results obtained, an average CV75 value of 24.9 μg/mL was calculated. This value was used for setting the concentration-range for measuring CD86 and CD54 expression in the main test. Due to the controversial findings on cytotoxicity obtained in the preliminary test, ten test concentrations instead of eight concentrations between 43.1 μg/mL and 8.3 μg/mL (nominal concentrations) were used for the main test in three independent experiments. All three experiments fulfilled the validity criteria and the study is therefore evaluated as valid. The increase in CD86 marker expression (RFI) was greater than 150 % at some tested doses (with > 50 % of cell viability) compared to the respective negative controls in all three independent and valid experiments. Based on these three valid positive responses, CD86 marker expression was concluded to be positive. Effective concentration for CD86 expression (EC 150) was not determined, since no clear dose response could be observed. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the respective negative controls at the lowest three tested concentrations (with > 50 % of cell viability) in all three independent and valid experiments. Based on the concordant results of the three valid individual runs for CD54 expression, the prediction was concluded positive. Effective concentration for CD54 expression (EC 200) was not determined, since no clear dose response could be observed. Since both CD86 and CD54 marker expressions gave positive result, the overall h-CLAT prediction was concluded to be positive.

Based on these results and the h-CLAT prediction model, the test item is concluded positive for skin sensitisation potential under the experimental conditions of human Cell Line Activation Test.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-04-13 to 2021-05-17

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:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in ultrapure water at a concentration of 200 mM.
- Preparation of the test chemical serial dilutions: Based on the test item stock solutions, 2-fold serial dilutions were made using the exposure medium to obtain twelve 100 × master concentrations of the test item creating a 100 × master plate. The 100 × master concentrations were further diluted 25- fold into exposure medium to obtain the 4 × master plate, by adding 10 μL of the 100 × master concentrations to 230 μL exposure medium and an extra 10 μL of DMSO to correspond to the same concentration of DMSO used in the positive control. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 μL of 4 × master solution to 150 μL of exposure medium).
- Preparation of the positive controls: The positive control used was trans-cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 μM.
- Preparation of the solvent, vehicle and negative controls: The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates. This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO used in the tested chemical and in the positive control.
- Stable dispersion obtained: A transparent, homogenous test item stock solution was obtained

DOSE RANGE FINDING ASSAY:
- Highest concentration used: No range-finding study was conducted. Final test concentrations ranged from 0.98 to 2000 μM (highest concentration recommended in OECD guideline 442D).
- Solubility in solvents: The test item was fully soluble in water, thus ultrapure water was used as a solvent instead of DMSO.
- Solubility in incubation medium: No precipitation was observed at any point during the assay.
- Cytotoxicity assessment performed: Yes. For the cell viability assay, medium was replaced after the 48-hour exposure time with MTT working solution (200 μL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 μL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: For each individual test, four parallel plates were used: three replicates were used for the luciferase activity induction measurements and one was needed for the MTT cell viability assay to measure the cytotoxicity induced by the test item.
- Number of repetitions: Two independent experiments were conducted.
- Test chemical concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM
- Application procedure: One day prior to testing cells were harvested in thawing medium and distributed into 96-well plates (10,000 cells/well) homogenously. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2. After the 24-hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test item and control substances were added to each well in a way that an additional 4-fold dilution was achieved on the plate for the final concentrations to be established (50 μL of 4 × master solution to 150 μL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test item.
- Exposure time: 48 ± 1 hours
- Study evaluation and decision criteria used: See "Any other information on materials and methods incl. tables"
- Description on study acceptance criteria: See "Any other information on materials and methods incl. tables"

SEEDING AND INCUBATION
- Seeding conditions: One day prior to testing, cells were harvested in thawing medium and distributed into 96-well plates (10,000 cells/well). Cells were grown for 24 ± 0.5 hours in 96-wells
microplates at 37 ± 1°C in the presence of 5 % CO2. The passage number at the start of the experiment was 9 and 11 at the end of the experiment.
- Incubation conditions: At 37 ± 1°C in the presence of 5 % CO2
- Washing conditions: After treatment, cells were washed with DPBS (270 μL).
- Precipitation noted: No

LUCIFERASE ACTIVITY MEASUREMENTS
- Choice of luminometer: Varioskan™ LUX Type 3020 with a Xenon flash lamp (100Hz) and a photodiode detector (wavelength range: 200 – 1000 nm). Luminescence was measured using a LAT module with a photomultiplier tube detector and a wavelength range of 360 - 670 nm.
- Plate used: 96-well plates
- Lysate preparation: After washing with DPBS (270 μL), 1 × lysis buffer (20 μL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 μL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

DATA EVALUATION
- Cytotoxicity assessment: Medium was replaced after the 48-hour exposure time with MTT working solution (200 μL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 μL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer. For calculations, see "Any other information on materials and methods incl. tables".
- Prediction model used: see "Any other information on materials and methods incl. tables".

Vehicle / solvent control:

water

Negative control:

other: DMSO

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. The EC1.5 values of the positive control fell between 7 μM and 30 μM (20 μM for both tests). The average inductions in the parallel plates for Trans-Cinnamaldehyde at 64 μM were 2.0 fold and 4.6 fold in the first and second test, respectively. In any of the tests, there was no cytotoxicity (or cell viability lower than 70 %) induced by the positive control at any of the tested concentrations.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

12.2

Cell viability:

Overall IC30 or IC50 values were determined as 342 μM and 394 μM respectively.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

394 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

342 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

36 µM

Cell viability:

Overall IC30 or IC50 values were determined as 342 μM and 394 μM respectively.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the test method described in the OECD Test Guideline 442D, the laboratory demonstrated technical proficiency using the 10 Proficiency Substances listed in APPENDIX IA - ANNEX 1 of TG 442D. Moreover, a historical database of data generated with the positive control is maintained over time to confirm the reproducibility of the test method in the laboratory

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes:
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

study cannot be used for classification

Remarks:

The KeratinoSens™ prediction must be considered in the framework of an IATA for a final prediction.

Conclusions:

In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method) according to OECD guideline 442D, the test item was concluded positive for skin sensitization potential under the experimental conditions of KeratinoSens™ method.

Executive summary:

In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) in accordance with OECD test guideline 442D and GLP. In order to derive a prediction for the test item the results of two independent tests were performed . Since the results of the two tests were concordant, a third test was not needed in order to derive a valid conclusion. The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests. In addition, all validity criteria for the negative and positive control were met. Thus, the assay is considered valid. For the test item, twelve doses ranging from 2000.00 μM to 0.98 μM were used in both tests. The test item induced cytotoxicity (a substance is considered cytotoxic if the cell viability falls below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Overall IC30 or IC50 values were determined as 342 μM and 394 μM respectively. Both valid tests were concluded positive, meaning that the induction values of the test item exceeded the 1.5-fold threshold. Therefore EC1.5 values were also determined, and the overall EC1.5 value was 36 μM. A clear dose response could be observed throughout the study. Based on these results and the KeratinoSens™ prediction model, the test item is concluded positive for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation in chemico, DPRA, RL1

In the course of this study the skin sensitization potential of the test item was studied using the Direct Peptide Reactivity Assay (DPRA) in accordance with OECD 442C and GLP. For the test item and the positive control substance, in order to derive a prediction two independent tests were evaluated, one with cysteine and one with lysine peptides. The results of two valid runs is used for the classification of the test item. Peptide depletion by the positive control cinnamaldehyde was 67.22 % ± 0.51 % for cysteine peptides and 52.07 % ± 1.33 % for lysine peptides, fulfilling all acceptance criteria for the positive control. The mean back-calculated peptide concentrations of the reference control A replicates were within the expected molarity concentration range for cysteine (0.49 mM) and lysine peptides (0.50 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.9 % and 0.2 % percentages for cysteine and lysine peptides, respectively. Moreover, the mean back-calculated peptide concentrations of the three reference controls C in the ultrapure water replicates were within the expected molarity concentration range for cysteine (0.47 mM) and lysine peptides (0.50 mM). For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 1.24 % ± 2.72 % while the percent lysine peptide depletion was 1.88 % ± 0.46 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 1.56 %, which is well below the threshold of 6.38 % for the applicable prediction model. The test item fells therefore into the no or minimal reactivity class and is evaluated not to be a skin sensitizer in this DPRA test. Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item showed no or minimal reactivity towards the synthetic peptides; thus it is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method according to OECD 442C.

 

Skin sensitisation in vitro, ARE-Nrf2, RL1

In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) in accordance with OECD test guideline 442D and GLP. In order to derive a prediction for the test item the results of two independent tests were performed . Since the results of the two tests were concordant, a third test was not needed in order to derive a valid conclusion. The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests. In addition, all validity criteria for the negative and positive control were met. Thus, the assay is considered valid. For the test item, twelve doses ranging from 2000.00 μM to 0.98 μM were used in both tests. The test item induced cytotoxicity (a substance is considered cytotoxic if the cell viability falls below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle. Overall IC30 or IC50 values were determined as 342 μM and 394 μM respectively. Both valid tests were concluded positive, meaning that the induction values of the test item exceeded the 1.5-fold threshold. Therefore EC1.5 values were also determined, and the overall EC1.5 value was 36 μM. A clear dose response could be observed throughout the study. Based on these results and the KeratinoSens™ prediction model, the test item is concluded positive for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

 

Skin sensitisation in vitro, h-CLAT, RL1

In the course of this in vitro human Cell Line Activation Test (h-CLAT) according to OECD 442E and GLP, the skin sensitisation potential of the test item was examined. The extent of cytotoxicity induced on THP-1 cells by the test item to allow a concentrationsetting for the main study was investigated in four dose finding tests. The maximal finalconcentrations used on the plates for the test item previously dissolved in saline were1000.0 μg/mL (first run), 43.0 μg/mL (second run), 63.0 μg/mL (third run) and 63.0 μg/mL (fourth run). Based on the results obtained, an average CV75 value of 24.9 μg/mL was calculated. This value was used for setting the concentration-range for measuring CD86 and CD54 expression in the main test. Due to the controversial findings on cytotoxicity obtained in the preliminary test, ten test concentrations instead of eight concentrations between 43.1 μg/mL and 8.3 μg/mL (nominal concentrations) were used for the main test in three independent experiments. All three experiments fulfilled the validity criteria and the study is therefore evaluated as valid. The increase in CD86 marker expression (RFI) was greater than 150 % at some tested doses (with > 50 % of cell viability) compared to the respective negative controls in all three independent and valid experiments. Based on these three valid positive responses, CD86 marker expression was concluded to be positive. Effective concentration for CD86 expression (EC 150) was not determined, since no clear dose response could be observed. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the respective negative controls at the lowest three tested concentrations (with > 50 % of cell viability) in all three independent and valid experiments. Based on the concordant results of the three valid individual runs for CD54 expression, the prediction was concluded positive. Effective concentration for CD54 expression (EC 200) was not determined, since no clear dose response could be observed. Since both CD86 and CD54 marker expressions gave positive result, the overall h-CLAT prediction was concluded to be positive. Based on these results and the h-CLAT prediction model, the test item is concluded positive for skin sensitisation potential under the experimental conditions of human Cell Line Activation Test.

 

DASS Automated Workflow OECD QSAR Toolbox (v4.5) prediction

The skin sensitising potential of the test item was calculated using the DASS Automated Workflow as part of the OECD QSAR Toolbox (v4.5).

Using the DASS Automated Workflow, the test item was calculated to be skin sensitising. The substance is within the applicability domain of the model. Thus the estimation is considered to be accurate.

 

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

 

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was described using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

 

 

Overall conclusion: In an integrated approach adressing key events of the skin sensitisation AOP, results from an in chemico skin sensitisation study according to OECD guideline 442C (DPRA, negative), from an in vitro ARE-Nrf2 Luciferase Test according to OECD guideline 442D (KeratinoSens, positive) and an in vitro human Cell Line Activation Test according to OECD guideline 442E (h-CLAT, positive) were combined and predicted a skin sensitising potential of the test item.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is classified and labelled for skin sensitisation, category 1B according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.