3-(2-methoxy-5-methylphenyl)-3-phenylpropanoic acid

Endpoint summary

Description of key information

Skin sensitisation: Weight of evidence. Based on the available data, the test item should be classified as Skin Sensitizer Category 1, according to Regulation (EC) No 1272/2008 (CLP). Three studies were conducted as part of an integrated approach (IATA) to support the identification of the sensitisation potential of the test item:

- In chemico skin sensitisation: Test method according to OECD 442C, GLP study. The test item showed Cysteine peptide depletion value of 1.14% and Lysine peptide depletion value of 0.20% i.e. an overall average of 0.67% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA. Therefore, the test item showed no skin sensitisation potential: a negative prediction for the first key event of the skin sensitisation AOP.

- In vitro skin sensitisation: Test method according to OECD 442D, GLP study. The test item presented negative results in two concordant runs, with an Imax = 1.18 (mean of two runs), and cell viability was > 70% in all runs for KeratinoSens™. Therefore, the test item showed no sensitisation potential: negative prediction for the second key event of the skin sensitisation AOP.

- In vitro skin sensitisation: Test method according to OECD 442E, GLP study. The test item presented positive results with RFI

of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of two concordant runs (and cell viability > 50%). Therefore, the test item showed skin sensitisation potential: positive prediction for the third key event of the skin sensitisation AOP. Based on a worst-case scenario approach, the test item is considered sensitising to the skin.

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:

From July 9th, 2018 to July 12th, 2018

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:

EURL ECVAM DB-ALM Protocol n.º 154

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

TEST SYSTEM
- Cysteine peptide (Ac-RFAACAA-COOH): source RS Synthesis, LLC, lot no. 170622
- Lysine peptide (Ac-RFAAKAA-COOH): source RS Synthesis, LLC, lot no. 170705
- Solvent/vehicle: acetonitrile, based on the results of the preliminary solubility test.
- Preparation of test item stock solution: the test item was dissolved at 100 mM in acetonitrile without sonication.
- Preparation of test item samples for the reactivity with cysteine peptide: 50 µL of test item formulation was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5 ± 0.05) and 200 µL of acetonitrile.
- Preparation of test item samples for the reactivity with lysine peptide: 250 µL of test item formulation was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2 ± 0.05).

CONTROLS (preparation)
- Positive control: 100 mM cinnamaldehyde (purity ≥ 95%, Sigma-Aldrich, lot no. MKBT8955V).
- Positive control for cysteine peptide: 50 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of cysteine peptide solution (at 0.667 mM in phosphate buffer at pH 7.5) and 200 µL of acetonitrile.
- Positive control for lysine peptide: 250 µL of cinnamaldehyde at 100 mM in acetonitrile was incubated with 750 µL of lysine peptide solution (at 0.667 mM in ammonium acetate at pH 10.2).
- Reference controls: All the reference control samples were prepared in triplicate at the nominal concentration of 0.500 mM of peptide in the solvent specified below.
- Reference control A: acetonitrile (to check calibration curve accuracy)
- Reference Control B: acetonitrile (included at the beginning and at the end of the sequence, to check the stability of the peptide over time)
- Reference Control C: acetonitrile (solvent used both for the test item and the positive control, to check the influence of the test item solvent on the peptide stability)
- Co-elution controls: 100 mM test item in the appropriate buffer.
- Co-elution control (cys p.): 50 µL test item formulation was incubated with 750 µL of cysteine peptide dilution buffer (without cysteine peptide) and 200 µL acetonitrile.
- Co-elution control (lys p.): 250 µL of test item formulation was incubated with 750 µL of lysine peptide dilution buffer (without lysine peptide).

HPLC ANALYSIS
- Equipment: HPLC system (Waters e2695) with autosampler, UV detector (200 nm; Waters 2489), Xbridge Peptide BEH C18 (100 x 2.1 mm; 3.5 µm) HPLC analytical column.
- Conditions: sample temperature 25ºC, column temperature 30ºC, injection volume: 7 µL(cys) or 3 µL(lys), flow rate 0.35 mL/min, total analysis time 20 min.
- mobile phase A: trifluoroacetic acid at 0.1% (v/v) in water
- mobile phase B: trifluoroacetic acid at 0.085% (v/v) in acetonitrile

- System suitability: calibration linearity: r2 > 0.9995 for both peptides; mean peptide concentration of Reference control A = 0.503 (cys) and 0.491 (lys) = 0.5 ± 0.005 mM.
- Analysis sequence: The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions). The reference controls C were placed at the beginning of each repetition. The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence. Lysine and cysteine analysis were conducted on separate day and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours.

ACCEPTANCE CRITERIA
- For the positive control, the mean % peptide depletion value must fall within 60.8 - 100.0% (cys) and 40.2 - 69.4 (lys);
- For the positive control, SD (cys) < 14.9% and SD (lys) < 11.6%;
- For the reference controls, CV% of the peak areas for reference controls B, C must be < 15.0%;
- For the reference controls in the analysis sequence, the mean peptide concentration of Reference control C must be 0.5 ± 0.005 mM;
- For the test item replicates, SD (cys) < 14.9% and SD (lys) < 11.6%;

INTERPRETATION OF RESULTS: Cysteine 1:10-only prediction model.
- 0.00 % ≤ mean % depletion ≤ 6.38 % = No or minimal reactivity = Negative DPRA Prediction
- 6.38 % ≤ mean of cysteine and lysine % depletion ≤ 22.62 % = Low reactivity = Positive DPRA Prediction
- 22.62 % ≤ mean of cysteine and lysine % depletion ≤ 42.47 % = Moderate reactivity = Positive DPRA Prediction
- 42.47 % ≤ mean of cysteine and lysine % depletion ≤ 100 % = High reactivity = Positive DPRA Prediction

Positive control results:

The depletion mean of cinnamaldehyde was 49.78 for lysine peptide and 78.17 for cysteine.

Key result

Run / experiment:

mean

Parameter:

other: cysteine peptide depletion (%)

Value:

1.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no or minimal peptide reactivity

Key result

Run / experiment:

mean

Parameter:

other: lysine peptide depletion (%)

Value:

0.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: no or minimal reactivity

Key result

Run / experiment:

mean

Parameter:

other: overall average

Value:

0.67

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:

OTHER EFFECTS:
- Appearance of precipitate (if yes, if precipitate was re-solubilised or centrifuged): no precipitate was observed.
- Co-elution: analysis of the chromatograms of the co-elution samples indicated that the test item did not co-elute with the cysteine or lysine peptides.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the expected DPRA prediction for the 10 proficiency substances was obtained. The resulted cysteine and lysine depletion values fall within the respective reference range for 8 out of the 10 proficiency substances for each peptide (8 out of 10 expected in OECD guideline).

ACCEPTANCE OF RESULTS:
The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied.
- Acceptance criteria met for reference controls: yes, the mean peptide concentrations of the reference control C samples was 0.506 mM (cys) and 0.482 mM (lys), within ± 10% of the nominal concentration; and the CV of the mean peptide peak area of the nine reference control B and C samples in acetonitrile was 0.64% (cys) and 1.02% (lys).
- Acceptance criteria met for positive control: yes , for cysteine peptide, the mean percent depletion value was 78.17%, within the acceptance range (60.8 - 100%, SD < 14.9%); and for lysine peptide, the mean percent depletion value was 49.78%, within the acceptance range (40.2 - 69.4, SD < 11.6%).
- Acceptance criteria met for variability between replicate measurements: yes, the maximum SD for the test item replicates was 1.14 < 11.6% for the percent cysteine depletion value, and 0.20 < 14.9%.

Table 1. Test item results: Percent peptide depletion.

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	0	1.54
Repetition 2	0.45	0.36		Mean Depletion %
Repetition 3	0.14	1.53
Mean	0.20	1.14		0.67

SD (Standard Deviation)	0.23	0.68
SD Validity criteria	< 11.6%	< 14.9%

Table 2. Positive control results: percent peptide depletion.

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	46.11	77.34
Repetition 2	51.03	78.17
Repetition 3	52.20	79.00
Mean	49.78	78.17
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100

Table 3. Reference controls.

Lysine Peptide

Cysteine Peptide

	Concentration (mM)	Concentration (mM)		Concentration validity criteria (mM)
Reference A	0.491	0.503		0.500 ± 0.050
Reference C	0.482	0.506

 

	CV %	CV %		CV validity criteria
Reference B/C	1.02	0.64		< 15 %

Interpretation of results:

GHS criteria not met

Conclusions:

The test item presents a Cysteine peptide depletion percentage of 1.14% and Lysine peptide depletion percentage of 0.20% i.e. an overall average of 0.67% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.

Executive summary:

A Direct Peptide Reactivity Assay has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item. The method was performed according to OECD OECD 442C, under GLP. The aim of the study is to evaluate the reactivity of the test item in chemico by quantifying the depletion of synthetic heptapeptides containing either lysine or cysteine.

A preliminary solubility study was conducted for the test item, and based on the results, the test item was prepared in acetonitrile. Peptide solutions were incubated with 100 mM test item solution or 100 mM cinnamic aldehyde solution (positive control), at ratios of 1:10 for cysteine and 1:50 for lysine. Reference controls and co-elution controls were run in parallel. After 24h incubation at 25ºC, the residual peptide concentrations were evaluated by HPLC-UV (220 nm). The test item presents a Cysteine peptide depletion percentage of 1.14% and Lysine peptide depletion percentage of 0.20% i.e. an overall average of 0.67% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA. The acceptance criteria for the calibration curve samples, the reference and positive controls as well as for the study samples were satisfied. Based on the test results, the test item shows no sensitisation potential.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From June 18th, 2018 to July 06th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
- Cell line used, storage conditions and source: KeratinoSens™ (Givaudan), exempt of mycoplasm.
- Passage number and level of confluence of cells: Cells were used at passage 20 in repetition 1 and passage 24 in repetition 2.
- Cell counting method used for seeding prior to testing and measures taken to ensure homogeneous cell number distribution:
- Luminometer used: GloMax™ (Promega) 96-well plate luminometer.
- Number of repititions and replicates: two independent runs were performed, with three replicates each. Each repetition was performed on a different day with fresh stock solution.
- Test item concentrations: On the basis of a preliminary solubility test results, the test item was dissolved in DMSO at 200 mM and then serial dilutions were performed. Both runs were performed using concentrations of 0.98, 1.95, 3.91, 7.81, 15.63, 31.3, 63, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.
- Controls: 1% DMSO, 1% Non-heat inactivated foetal calf serum in culture medium was used as vehicle/negative control; cinnamic aldehyde (Sigma Aldrich lot no. MKBT8955V) was used for the positive control; cytotoxicity was measured by MTT reduction.
- Application procedure and exposure time: After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL treatment medium. Then, from the Master plate 4x, a volume of 50 µL was added to each well of the 3 assay plates and 50 µL to 2 plates for the cytotoxicity evaluation. All plates were covered by a sealing membrane to avoid evaporation and to avoid cross-contamination between wells, and the plates were then incubated for 48 (± 1) hours at 37ºC, 5% CO2, 90% humidity.
- Description of evaluation and decision criteria used: The results of each run are analyzed individually. The test item is considered as positive if the following 4 conditions are all met in 2/2 or in 2/3 runs, otherwise the KeratinoSens prediction is considered as negative:
- the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
- at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
- there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).
- Description of study acceptance criteria used: Each run was considered valid if the following criteria were met:
- the positive control results should be positive, thus gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
- the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of cinnamic aldehyde was carefully checked, and the run was accepted if there was a clear dose response with increasing luciferase activity at increasing concentrations for the positive control,
- the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

Positive control results:

Imax = 3.35 (mean); EC1.5 = 10.99 (geometric mean)

Key result

Run / experiment:

other: #1

Parameter:

other: Imax

Value:

3.8

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: #2

Parameter:

other: Imax

Value:

2.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: #1

Parameter:

other: Viability

Remarks:

IC70 (µM)

Value:

1 043.2

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: #2

Parameter:

other: Viability

Remarks:

IC70 (µM)

Value:

587.94

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Parameter:

other: EC1.5

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: recommended substances for demonstrating technical proficiency with the KeratinoSens™ test method were tested to validate the method.

ACCEPTANCE OF RESULTS:
- All acceptance criteria were fulfilled for the positive and negative controls in each run. These runs were therefore considered to be valid.
- Acceptance criteria met for negative control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% in each repetition.
- Acceptance criteria met for positive control: yes. A positive result was obtained; gene induction was statistically significant above the threshold of 1.5 in at least one of the tested concentrations; EC1.5 = 12.38 µM, value was within two standard deviations of the historical mean; Imax in the three replicate plates for the positive control at 64 µM was between 2 and 8.
- Acceptance criteria met for variability between replicate measurements: yes.
- Range of historical values if different from the ones specified in the test guideline: 3 µM ≤ EC1.5 ≤ 22 µM (n=233).

Positive control (*geometric mean).

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.19	1.28	1.78	2.35	3.80	11.52	3.80
Rep 2	1.19	1.38	1.78	2.26	2.90	10.48	2.90
Mean	1.19	1.33	1.78	2.31	3.35	10.99*	3.35

Solvent control.

Control solvent	CV %  control solvent
Rep 1	9.8
Rep 2	11.3

Test item.

	VIABILITY	INDUCTION
ID-18/05481	IC70  µM	Imax	Linear EC1.5  µM	EC1.5 Lin/Log  µM
Rep 1	1043.20	1.19	-	-
Rep 2	587.94	1.16	-	-
Mean	-	1.18	-	-
Geometric mean	783.16	-	-	-

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed an Imax = 1.18 (mean of two runs), and cell viability was > 70% in all runs. Therefore, the test item is not a skin sensitiser.

Executive summary:

A KeratinoSens™ study has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item. The method was performed according to OECD 442D, under GLP. The aim of the study is to evaluate the potential of the test item to activate the Nrf2 transcription factor by quantifying the luciferase induction after exposure of these keratinocytes to the test item for 48h. 12 concentrations of the test item ranging from 0.98 to 2000 μM were prepared by serial dilution in culture medium containing 1% DMSO and added to 96-well plates of KeratinoSens cells, in two separate runs of three replicates each. Positive and negative controls were run in parallel, as well as a cytotoxicity assay (MTT reduction). All acceptance criteria were fulfilled for the positive and negative controls in each run. Under the experimental conditions described, the test item presented negative results in both runs, with a cell viability > 70% and and Imax 1.19 and 1.16 for the first and second runs, respectively. No geometric mean IC30 or IC50 were calculated since the cell viability was > 70% in both runs. Based on the test results, the test item shows no sensitisation potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From November 27th, 2018 to February 5th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD guideline No. 442E: "In vitro skin sensitization: human Cell Line Activation Test (h CLAT)"

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
TEST SYSTEM
- Cell line used, storage conditions and source: THP-1 (ATCC, #TIB-202), stored in liquid nitrogen and sub-cultured twice weekly.
- Culture medium and conditions: RPMI 1640 medium, GlutaMAX™ supplement includinc 25 mM HEPES, supplemented with 10% FBS, 0.05 mM 2-mercapt
oethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin).
The cells were grown using general culture procedures and maintained in a humidified incubator set at 37± 1.5ºC, 5± 0.5% CO2 and were not allowed to exceed a cell density of 1 E06 cells/mL or more than 30 passages. Reactivity checks were performed to qualify the cells before testing.
The passage numbers of the used THP-1 cells were 17 and 18 in the cytotoxicity tests and 18 and 21 in the h CLAT for runs 1 and 2, respectively.
- Cell culture for testing: On the day of the cytotoxicity or main experiment, directly before the treatment of the cells, a volume of 500 µL with a cell density of 1.8 - 2 E06 THP-1 cells/mL was seeded in each corresponding well of a 24-well flat bottom plate.

CONTROLS
- Solvent/vehicle control: dimethylsulfoxide (DMSO, purity ≥ 99%), dissolved at 0.2% in culture medium.
- Medium control: culture medium.
- Positive control: 2,4-Dinitrochlorobenzene (DNCB, purity ≥ 99%) at 3 and 4 µg/mL.

STUDY DESIGN
- Solubility assessment: Test item solutions were prepared in saline (0.9% NaCl) and DMSO. The solubility of the test item was assessed visually for each preparation (particles, drops, cloudiness, non-miscible phases, etc). Based on the results, 5000 µg/mL in culture medium was used as highest test item concentration in the cytotoxicity test.
- Dose Finding assay (PI Assay): For each of 2 independent cytotoxicity tests, 8 concentrations of test item were prepared by 1:2 serial dilutions using the selected vehicle, and sonicated for 5 minutes. 500 μL of the working solutions were added to the volume of THP-1 cell suspension in the
plate (500 μL) to achieve a further 2-fold dilution. The plates were incubated for 24 ± 0.5 h. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations (the 2 highest concentrations of both cytotoxicity tests could not be evaluated due to observed strong precipitation). Then, cells were transferred into sample tubes and collected by centrifugation, washed twice with 600 μL of FACS buffer (PBS with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added in each sample tube. Then, the flow cytometer (FACSCalibur, Becton Dickinson GmbH) was calibrated and the cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal. The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.
- Main test (CD86/CD54 expression measurement). 2 independent validated runs were conducted on different days. The highest concentration corresponded to 1.2-fold the mean CV75. Based on the previous tests, the following concentrations of test item were used in the main test: 117, 141, 169, 203, 243, 292, 350 and 420 µg/mL. Exposure was carried out as in the Dose Finding assay. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations. Then cells were centrifuged, re-suspended and blocked with 600 µL of blocking solution at 2-8ºC (on ice) for approximately 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2 - 8ºC or on ice during the staining and analysis procedures. Those cells were mixed and incubated for 30 ± 5 min. at 2 - 8ºC, protected from light. The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry as in the DRF. Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis.

INTERPRETATION OF RESULTS
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs:
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
− Otherwise, the h-CLAT prediction is considered NEGATIVE.

ACCEPTANCE CRITERIA
- Cell viability of medium control and DMSO control should be more than 90%.
- In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
- For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
- Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. If 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90%.

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%, with one exception. The CD54 RFI value of the positive control (4.0 µg/mL DNCB) in the second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%) due to a technical error. However, this one exception is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the second h-CLAT run exceeded the positive criteria.

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Precipitation:
1) DRF test: The two highest concentrations of both cytotoxicity tests could not be microscopically evaluated due to observed strong precipitation.
2) Main test: No precipitation was observed at any dose tested.

ACCEPTANCE OF RESULTS: All acceptance criteria were met in each run (see 'overall remarks').

DEMONSTRATION OF TECHNICAL PROFICIENCY: The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated. The h-CLAT proficiency was conducted using XTT for dose range finding. The cell viability in the main experiments was calculated using the mean of the GeoMean(7-AAD) isotype control, GeoMean(7-AAD) CD54 and GeoMean(7-AAD) CD86.

Dose-Range Finding results:

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 625 µg/mL in both cytotoxicity tests (threshold: < 75%). The mean CV75 value of both cytotoxicity tests was 349.94µg/mL. Based on the results from both DRF runs, The following concentrations of the test item were tested in the main experiments (h-CLAT): 117, 141, 169, 203, 243, 292, 350 and 420 µg/mL.

Main test

Table 3. Results of the first h-CLAT run for the Test Item.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	92.34
DMSO Control	-	100.0	100.0	92.59
Positive Control (DNCB)	3.0	284.1*	363.7*	78.54
	4.0	286.4*	345.4*	76.07
Test Item	117	94.1	140.6	91.71
	141	235.3*	169.1*	83.22
	169	188.2	171.8*	81.03
	203	225.5*	144.1	82.67
	243	84.3	179.2*	80.27
	292	141.2	341.7*	63.19
	350	-39.2	483.1*	47.37
	420	211.8	490.5*	44.77

*: RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

- shade: cell viability below 50%, values excluded from the evaluation.

Table 4. Results of the first h-CLAT run for the Test Item.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	90.81
DMSO Control	-	100.0	100.0	91.99
Positive Control (DNCB)	3.0	218.8*	200.0*	79.86
	4.0	127.6#	379.2*	65.61
Test Item	117	202.7*	175.9*	86.47
	141	162.2	98.5	86.92
	169	171.1	145.6	82.35
	203	152.0	151.8*	79.66
	243	129.8	177.2*	86.11
	292	171.6	163.3*	85.26
	350	121.3	197.7*	70.80
	420	94.7	217.9*	64.57

*: RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

#: CD54 RFI value of the positive control (4.0 µg/mL DNCB) did not fulfil the positive criterion (CD54 ≥ 200%) due to a technical error. However, this one exception is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the second h-CLAT run exceeded the positive criteria.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

EU criteria.

Conclusions:

The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. Therefore, the test item activated THP-1 cells under the test conditions of this study and it is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

An in vitro Human Cell Line Activation Test (h-CLAT) was performed as part of an integrated approach to support the identification of the sensitization potential of the test item in accordance with OECD Guideline 442E, under GLP conditions. Based on the results from a solubility assay two Dose-Range Finding (cytotoxicity) assays, the upper dose tested was 420 µg/mL, using DMSO as a vehicle. Two validated successive test runs were performed. In each run, the test item formulations (117, 141, 169, 203, 243, 292, 350 and 420 µg/mL) were applied to THP-1 cells and cultured for 24 hours ± 0.5 hours at 37ºC, 5% CO2. Negative (medium), vehicle (DMSO), and positive (DCNB) controls were run in parallel. After incubation, the expression of CD86 and CD54 was measured by flow cytometry, and the viability of the cells was determined after staining with 7-Aminoactinomycin D (for which demonstration of technical proficiency had been performed). The Mean Fluorescence Intensity (MFI) was obtained for each test sample and then corrected. The corrected MFI values were compared to the corresponding vehicle control to obtain the Relative Fluorescence Index (RFI) for CD86 and CD54 expression. All validity criteria were met. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. A slight dose response could be observed for CD86 in both h-CLAT runs. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information (negative results for OECD 442C, 442D tests, but positive result for OECD 442E test) the substance is proposed to be classified as Skin sensitiser Category 1, according to CLP Regulation (EC) No. 1272/2008.