Dibenzoyl-L-tartaric acid monohydrate

Endpoint summary

Description of key information

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 a mean depletion value of 4.03% in a first run and 1.07% in a confirmatory run, reflecting no or minimal reactivity according to test criteria. 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 a cell viability > 70% and Imax values of 1.33, 1.81 and 1.21 for each of three independent runs (mean value 1.34) for KeratinoSens™, respectively. 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(CD86) > 150% in two concordant runs, with a 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:

05/11/2018 - 20/02/2019

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)

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 chemico test system) - Details on study design:

TEST SYSTEM
- Cysteine peptide (Ac-RFAACAA-COOH): source RS Synthesis, LLC, purity > 90%
- Lysine peptide (Ac-RFAAKAA-COOH): source RS Synthesis, LLC, purity > 90%
- 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) 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).

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 ± 0.05) 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 ± 0.05).
- 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 (to check the stability of the peptide over time)
- Reference Control C: acetonitrile (solvent used both for the test item and the positive control)
- 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 with autosampler, UV detector (200 nm), Zorbax SB 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 > 0.990 for both peptides; mean peptide concentration of Reference control A = 0.508 (cys) and 0.498 (lys) = 0.5 ± 0.005 mM.
- Analysis sequence: For each peptide, the analytical sequence included at least: one blank sample (peptide dilution buffer), one calibration curve injected at the beginning of the analytical batch, three reference control A samples, the co-elution control sample, three reference control B samples, reference control C samples (replicate 1), positive control sample (replicate 1), and test item study sample (replicate 1).

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 53.13 for lysine peptide and 74.43 for cysteine.

Key result

Run / experiment:

other: mean / run # 1

Parameter:

other: cysteine depletion (%)

Value:

5.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: mean / run # 1

Parameter:

other: lysine depletion (%)

Value:

2.92

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: mean / run # 2

Parameter:

other: cysteine depletion (%)

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: mean / run # 2

Parameter:

other: lysine depletion (%)

Value:

2.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- The test item showed a mean depletion of 5.14% for Lysine and 2.92% for Cysteine, i.e. an overall average of 4.03% reflecting no reactivity and thus a negative prediction of DPRA. However, as the mean percent depletion falls in the range 3% - 10% for the cysteine 1:10 / lysine 1:50 prediction model, a second run was considered. In the second run, the test item showed a mean depletion of 2.14% for Lysine and 0% for Cysteine, i.e. an overall average of 1.07% reflecting no reactivity and thus confirming the negative DPRA prediction.
- 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 (as recommended by the OECD TG).

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. First run: the mean peptide concentrations of the reference control C samples was 0.505 mM (lys) and 0.491 mM (cys), 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% (lys) and 1.09% (cys). Second run: the mean peptide concentrations of the reference control C samples was 0.539 mM (lys) and 0.486 mM (cys), 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 2.20% (lys) and 2.13% (cys).
- Acceptance criteria met for positive control: yes. First run: for cysteine peptide, the mean percent depletion value was 74.43%, within the acceptance range (60.8 - 100%); and for lysine peptide, the mean percent depletion value was 53.13%, within the acceptance range (40.2 - 69.4). Second run: for cysteine peptide, the mean percent depletion value was 68.97%, within the acceptance range (60.8 - 100%); and for lysine peptide, the mean percent depletion value was 55.44%, within the acceptance range (40.2 - 69.4).
- Acceptance criteria met for variability between replicate measurements: yes, the maximum SD for the test item replicates was < 11.6% for the percent lysine depletion value, and < 14.9% for the percent cysteine depletion in both runs.

Table 1. Run 1: Test item results.

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	4.46	2.65
Repetition 2	5.21	1.99
Repetition 3	5.75	4.13
Mean	5.14	2.92
Mean depletion %	4.03
SD	0.65	1.10
SD Validity criteria	< 11.6%	< 14.9%

Table 2. Run 1: Positive controls.

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	51.26	74.95
Repetition 2	54.80	74.88
Repetition 3	53.33	73.47
Mean	53.13	74.43
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100

 

Table 3. Run 1: Reference controls.

	Lysine Peptide	Cysteine Peptide	Concentration validity criteria (mM)
	Concentration (mM)	Concentration (mM)
Reference A	0.501	0.487	0.500 ± 0.050
* Reference C	0.505	0.491

	CV %	CV %	CV validity criteria
Reference B/C	0.64	1.09	< 15 %

Table 4. Run 2: Test item.

	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	4.92	0
Repetition 2	1.51	0
Repetition 3	0	0
Mean	2.14	0
Mean depletion %	1.07
SD	2.52	0
SD Validity criteria	< 11.6%	< 14.9%

Table 5. Run 2: Positive controls.

Cinnamaldehyde	Depletion in Lysine Peptide %	Depletion in Cysteine Peptide %
Repetition 1	55.70	68.04
Repetition 2	55.19	69.01
Repetition 3	55.43	69.87
Mean	55.44	68.97
Depletion Validity criteria	40.2 < Depletion < 69.4	60.8 < Depletion < 100

Table 6. Run 2: Reference controls.

	Lysine Peptide	Cysteine Peptide	Concentration validity criteria (mM)
	Concentration (mM)	Concentration (mM)
Reference A	0.529	0.496	0.500 ± 0.050
* Reference C	0.539	0.486

	CV %	CV %	CV validity criteria
Reference B/C	2.20	2.13	< 15 %

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria.

Conclusions:

The test item showed mean % depletion of 5.14% for Lysine and 2.92% for Cysteine in a first run and 2.14% for Lysine and 0% for Cysteine in a confirmatory run. Based on these results, the DPRA prediction was negative. The test item showed no sensitisation potential under test conditions.

Executive summary:

A Direct Peptide Reactivity Assay was 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). Under test conditions, no precipitation or co-elution was observed; all the validity criteria were met. In a first run, the test item showed a mean depletion of 5.14% for Lysine and 2.92% for Cysteine, i.e. an overall average of 4.03% reflecting no reactivity and thus a negative prediction of DPRA. However, as the mean percent depletion falls in the range 3% - 10% for the cysteine 1:10 / lysine 1:50 prediction model, a confirmatory run was considered. In the second run, the test item showed a mean depletion of 2.14% for Lysine and 0% for Cysteine, i.e. an overall average of 1.07% reflecting no reactivity and thus confirming the negative DPRA prediction. Based on the test results, the test item shows no sensitisation potential under test conditions.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19/11/2018 - 25/01/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 25th, 2018.
EURL ECVAM DB-ALM Protocol n.º 155

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

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:
- Cell line used, storage conditions and source: KeratinoSens™ (Givaudan), exempt of mycoplasm; frozen.
- Passage number and level of confluence of cells: cells were grown using general culture procedures up to 80-90% confluence. In order to ensure a good cell concentration, cultures were split to a ratio of 1/6 for 3 days or 1/12 for 4 days. Cells were used at passage 17, 19 and 18 in runs 1, 2, 3, respectively.
- Cell counting method used for seeding prior to testing and measures taken to ensure homogeneous cell number distribution: Malassez cell hemocytometer.
- Luminometer used: GloMax™ (Promega) MULTISKAN EX plate reader (Thermo life sciences) for 96-well plates. Reading range 0 - 3.5 units of Absorbance; linearity range 0 - 2.200 units of Absorbance.
- Number of repititions and replicates: 3 independent runs were performed, with 3 replicates each. New formulations were prepared for each run.
- Test item concentrations: On the basis of a preliminary solubility test results, the test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
- Controls: 1% DMSO in culture medium was used as vehicle/negative control; cinnamaldehyde was used for the positive control (5 concentrations from 4 to 64 µM according to a geometric progression of ratio 2); cell viability was assessed with the MTT method.
- Application procedure and exposure time: After incubation of the seeded plates for 24 ± 1 hours at 37ºC, 5% CO2, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of three white plates (assay plates) and in two transparent plates (cell viability assessment). All plates were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 ± 1 hours (37ºC, 5% CO2, 90% humidity).
- Description of evaluation and decision criteria used: The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. If the first two runs were not concordant, a third run is performed and the final outcome is that of the two concordant runs. The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or in 2 of 3 repetitions. 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),
- the EC1.5 value is < 1000 µM,
- at the lowest concentration with a gene induction ≥ 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
- there is an apparent overall dose-response for luciferase induction, which is similar between repetitions.
- Description of study acceptance criteria used: Each run was considered valid if the following criteria were met:
- in the the positive controls, gene induction must 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 and the average induction (Imax) in each repetition for cinnamaldehyde 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,
- in the negative control, the average coefficient of variation of the luminescence reading of the triplicate plates (3x6 wells) should be < 20% in each repetition. If for one repetition the validity criteria are not met, or in case of equivocal result additional repetitions should be considered.

Positive control results:

Imax = 4.96 (geometric mean); the results for each run were: 5.11 (run 1), 4.52 (run 2), 5.25 (run 3).

Key result

Run / experiment:

other: #1

Parameter:

other: Imax

Value:

1.33

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:

1.81

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: inconclusive

Remarks:

only one concentration (500 µM) is > 1.5 and statistically significant compared to the negative control. No dose effect is observed.

Key result

Run / experiment:

other: #3

Parameter:

other: Imax

Value:

1.21

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: #1, 2, 3

Parameter:

other: cell viability

Value:

2 000

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: #2

Parameter:

other: EC1.5

Value:

360.9

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no.
- No precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period.
- No noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), IC30 and IC50 was > 2000 in all runs,
- the Imax mean value was < 1.5 (i.e. 1.45).

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 validity 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/solvent control: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% in each repetition (i.e. 10.5, 11.2, 12.9).
- Acceptance criteria met for positive control: yes. Gene induction was statistically significant above the threshold of 1.5 in at least one of the tested concentrations; EC1.5 = 6.89 µM, value was within 2*SD 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: 2.5 µM ≤ EC1.5 ≤ 21 µM.

Table 1. Results for the positive control.

Cinnamaldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.44	1.55	2.09	3.13	5.11	6.15	5.11
Rep 2	1.33	1.47	2.00	2.96	4.52	8.49	4.52
Rep 3	1.27	1.68	2.06	3.34	5.25	6.25	5.25
Mean	1.34	1.57	2.05	3.14	4.96	6.89*	4.96

Table 2. Results for the negative control.

Control solvent	CV %  control solvent
Rep 1	10.5
Rep 2	11.2
Rep 3	12.9

Table 3. Results for the test item.

	VIABILITY		INDUCTION
ID-18/10028	IC50 µM	IC30 µM	Imax	Linear EC1.5  µM	EC1.5 Lin/Log  µM
Rep 1	>2000	>2000	1.33	-	-
Rep 2	>2000	>2000	1.81	382.42	360.90
Rep 3	>2000	>2000	1.21	-	-
Mean	-	-	1.45	-	-
Geometric mean	>2000	>2000	-	-	-

Interpretation of results:

GHS criteria not met

Remarks:

EU criteria.

Conclusions:

The test item showed an Imax = 1.45 (mean of 3 runs), and cell viability was > 70% in all runs. Based on this result, the test item showed no sensitisation potential under test conditions.

Executive summary:

A KeratinoSens™ study was 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 conditions. 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 3 separate runs of 3 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 2 concordant runs out of 3, with a cell viability > 70% in all runs (IC30 and IC50 > 2000 µM). The Imax values were 1.33, 1.81 and 1.21 for each run, respectively. Based on the test results, the test item shows no sensitisation potential under test conditions.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28/01/2019 - 21/02/2019

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-mercaptoethanol, 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 alt
erations (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: 1180, 1416, 1699, 2039, 2447, 2936, 3523 and 4228 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.

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (CD86)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: RFI (CD54)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Precipitation: no.

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:

Results obtained in the first DRF test (i.e. DRF 1):  The CV75 value of the first Cytotoxicity Test: 2692.52 µg/mL.

Results obtained in the second DRF test (i.e. DRF 2): Due to the lack of cytotoxicity in the flow cytometric evaluation of the cytotoxicity test no.2, a CV75 value could not be calculated. Therefore the highest tested test item concentration of 5000 µg/mL was used as CV75 value to calculate the mean CV75 for the main experiments.

Results obtained in the second DRF test (i.e. DRF 3):  The CV75 value of the third Cytotoxicity Test: 2877.58 µg/mL.

The mean CV75 value of all three Cytotoxicity Tests: 3523.37 µg/mL. Based on the results from the DRF runs, the maximum concentration tested in the main test was 4228 µg/mL.

Main test

Table 4. Main test individual results. Run 1.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	95.75
DMSO Control	-	100.0	100.0	96.24
Positive Control (DNCB)	3.0	343.0*	1952.4*	79.23
	4.0	650.6*	1672.8*	79.75
Test Item	1180	83.9	284.8*	96.13
	1416	80.6	289.9*	96.06
	1699	101.6	324.2*	96.07
	2039	100.0	287.9*	95.65
	2447	140.3	231.3*	96.51
	2936	117.7	224.2*	96.65
	3523	135.5	221.2*	96.36
	4228	141.9	251.5*	95.54

 

Table 5. Main test individual results. Run 2.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	93.91
DMSO Control	-	100.0	100.0	96.17
Positive Control (DNCB)	3.0	251.6*	609.2*	87.24
	4.0	338.5*	562.3*	85.59
Test Item	1180	89.2	83.1	95.82
	1416	93.7	110.7	95.64
	1699	100.9	113.2	94.51
	2039	112.6	116.6	90.84
	2447	112.6	106.8	96.04
	2936	119.8	111.0	95.45
	3523	111.7	115.5	94.02
	4228	125.2	351.0*	74.36

Interpretation of results:

other: indication of skin sensitising potential

Remarks:

EU criteria.

Conclusions:

The RFI of CD86 was greater than 150% in all tested concentrations of the first run and in the highest tested concentration of the second independent run. 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 to assess the dendritic cell activation potential 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 4228 μg/mL, using DMSO as a vehicle. Two validated successive test runs were performed. In each run, the test item formulations (1180, 1416, 1699, 2039, 2447, 2936, 3523 and 4228 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 was greater than 150% in all tested concentrations of the first run and in the highest tested concentration of the second independent run. No clear dose response could be observed. Based on the test results, 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.