N,N'-(methylenedi-p-phenylene)bis(aziridine-1-carboxamide)

Endpoint summary

Description of key information

First key event (molecular initiating event): Weight of evidence. Test method according to the OECD 442C Guideline with GLP. The test item cannot be classified as either “sensitizer” or “nonsensitizer” in the DPRA test since its elution was occurred at the same time as that of elution of Cysteine peptide.

Second key event (Activation of keratinocytes): Weight of evidence. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test substance may be classified as skin sensitizer using the KeratinoSensTM test method.

Third key event (activation of dendritic cells): Weight of evidence. Test method according to the OECD 442E Guideline with GLP. Under the experimental conditions the test substance may be classified as skin sensitizer using the h-CLAT test method.

Skin sensitisation: Weight of evidence. Integrated testing strategy (ITS). Based on the“2 out of 3-sens ITS” approach as described in annex 1 of OECD guidance ENV/JM/MONO(2016)29, two positive results obtained in the tests addressing two different key events means that the test substance does need to be classified as skin sensitizer.

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

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))

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:
Stock solutions of Cysteine (Ac-RFAACAA-COOH) and Lysine (Ac-RFAAKAA-COOH) were freshly prepared just before their incubation with the test item. The final concentration of the Cysteine peptide was 0.667 mM in pH 7.5 phosphate buffer whereas the final concentration of the Lysine peptide was 0.667 mM in pH 10.2 ammonium acetate buffer.
- Preparation of the test chemical solutions:
Test item was weighed into clean and sterile vials. Test item was dissolved in 3.0 mL of the 1:1 DMSO: acetonitrile to prepare a 100 mM solution immediately prior to use. The weight of test item to be added in the vial was determined based on the molecular weight (MW) and purity.
- Preparation of the positive controls, reference controls and co-elution controls:
Cinnamic aldehyde was used as positive control (PC) at a concentration of 100 mM in acetonitrile.
Reference control A was prepared with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.
Preparation of reference control A (0.5 mM): 750 µL Cysteine/Lysine peptide (0.667 mM) + 250 µL of acetonitrile
Reference control B was prepared with acetonitrile and its replicates were injected in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
Preparation of reference control B: Same as preparation for reference control A (but run before and after 24 ± 2 hours incubation at 25 ± 2.5 °C in dark).
Reference control C was prepared with the solvent used to solubilize the test items, i.e., 1:1 mixture of DMSO:Acetonitrile and included in every assay run together with the samples. They were used to verify that the solvent does not impact the Percent Peptide Depletion. An appropriate Reference Controls C for each test item was used to calculate Percent Peptide Depletion.
Preparation of reference control C: 750 µL of Cysteine peptide stock + 200 µL of acetonitrile + 50 µL 1:1 mixture of DMSO:Acetonitrile (for Cysteine). 750 µL of Lysine peptide stock + 250 µL of 1:1 mixture of DMSO:Acetonitrile (for Lysine).
Co-elution control was prepared without peptide, to verify whether the test item absorbs at 220 nm and has a similar retention time as a peptide and/or interfere with the data analysis.
Co-elution Control preparation: 750 µL of phosphate buffer (pH 7.5) + 200 µL of acetonitrile + 50 µL test item (Cysteine). 750 µL of ammonium acetate buffer (pH 10.2) + 250 µL of test item (Lysine).

INCUBATION
- Incubation conditions: Cysteine and Lysine peptide solutions were incubated in glass auto sampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5 ºC for 24 ± 2 hours before running the HPLC analysis. Test item was analysed in triplicate for both peptides.
- Precipitation noted: The test item was found to be insoluble in acetonitrile, Milli-Q water, isopropanol, acetone, 1:1 acetone: acetonitrile, and 1:9 dimethyl sulfoxide (DMSO): acetonitrile and found to be soluble in 1:1 DMSO: acetonitrile at the concentration of 100 mM. Precipitation was not observed at 100 mM.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: A standard calibration curve was generated on each day of HPLC analysis and value of r2 obtained was 0.99987 for Cysteine and 0.99909 for Lysine containing peptides. This showed that system was suitable for assay.

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

Vehicle / solvent:

mix DMSO:acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Values of the mean percent peptide depletion value of positive control cinnamic aldehyde was 76% for cysteine peptide and 45% for Lysine peptide.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

-122 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

(co-elution was observed with cysteine peptide)

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Mean of lysine and cysteine peptides

Remarks on result:

not determinable

Remarks:

(Mean percent depletion was not calculated since co-elution was observed with cysteine peptide)

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.50± 0.05 mM
- Acceptance criteria met for reference control B: yes, the CV (coefficient of variation) of the controls B was 1.60% for Cysteine peptide and 3.78% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.39 mM ((might be due to solvent). The CV (coefficient of variation) of the controls C was 0.66% for Cysteine peptide and 0.71% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for positive control: The depletion mean rate was 76% for cysteine peptide and 45% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.

Mean percent depletion for the test item was not calculated since co-elution was observed with cysteine peptide.

Interpretation of results:

other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.

Conclusions:

The test item cannot be classified as either “sensitizer” or “non-sensitizer” in the DPRA test since its elution was occurred at the same time as that of elution of Cysteine peptide.

Executive summary:

A DPRA skin sensitization test was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item was prepared at 100 mM in 1:1 DMSO: acetonitrile and Cinnamaldehyde (positive control) was prepared at 100 mM in acetonitrile. Reference controls A, B and C were prepared with acetonitrile and 1:1 DMSO: acetonitrile (control C) in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C±2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested in triplicate for both peptides. All validity criteria were fulfilled. Percent peptide depletion values of the test substance for Cysteine and Lysine were -122% (which shows significant co-elution) and 1%, respectively. Mean percent depletion was not calculated since co-elution was observed with cysteine peptide. Thus, the test substance cannot be classified as either “sensitizer” or “non-sensitizer” and hence the result of DPRA test is considered as “inconclusive”.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 January 2018 - 11 February 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 of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

Details on study design:

REAGENTS AND MEDIA
-Fetal Bovine Serum (FBS): Gibco (Lot # 1789459)
-DMSO: Qualigens (Lot # 2217110817)
-D-MEM with Glutamax: Gibco (Lot # 1897167)
-Ethylene Diamine Tetra Acetic Acid (EDTA): Sigma (Lot # SLBM9213V)
-DPBS (1X): Gibco (Lot # RNBF8606)
-Trypsin-EDTA: Gibco (Lot #1894157)
-Geneticin: Gibco(Lot #1751936)
-Culture medium: D-MEM with Glutamax supplemented with 9.1% fetal bovine serum and 500 μg/mL geneticin.
-Exposure medium: D-MEM supplemented with 1% foetal bovine serum (495 mL D-MEM + 5 mL FBS).
-Staining solution: MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in culture medium.
-Passage number: cells were used at passage 21 (experiment 1 and 2) and passage 24 (experiment 3).

CONTROLS
-Positive control: cinnamaldehyde (CAS 14371-10-9; batch no STBF4119V; purity 99.4%)
-Negative (solvent) control: DMSO

CELLS SEEDING.
-Cells suspension: Cells were maintained in culture medium. After cells attained 80-90% confluency, they were washed twice with DPBS containing 0.05% of EDTA. To each flask 2-3 mL of 0.05% Trypsin-EDTA was added and flasks were incubated at 37 ± 1ºC (usually 5–10 minutes). After cells were detached completely, they were resuspended in DMEM with 9.1% FBS without geneticin. Cell count was taken and cell density was adjusted to 8 x 10e4 cells/mL. 125 µL of this culture (containing approx. 10,000 cells) was then dispensed in each well of 96 well plates, leaving one cell empty (to assess background values).
-nº of culture plates: four; three 96-well white assay plates (for luciferase assay) and one 96-well flat bottom transparent plate (for cytotoxicity, MTT assay).
-Cell density: 10e4 cells per well.
-Incubation: the seeded plates were incubated for 24 hours in 5±1 % CO2 at 37±1ºC.

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Positive control: Trans cinnamaldehyde was dissolved in DMSO to achieve concentration of 200 mM. This solution was further diluted to final concentration of 6.4 mM by adding 32 µL of 200 mM solution to 968 µL of DMSO.
Negative control: DMSO was used as negative (solvent) control.
Test Item Preparation: A quantity of 40 mg of test item was dissolved in 1 mL DMSO to attain the stock solution concentration of 40 mg/mL.
Preparation of 100X Master Plate: A 100-fold concentrated dilutions series was prepared in 96-well plate:
-For Test Item Row (Row A): 100 µL of DMSO was added in column 1-11 of rows A. Then 100 µL of 40 mg/mL of stock solution of test item was added in column 11 & 12 of corresponding rows. Serial dilutions were prepared by transferring 100 µL from column 11 to column 10 and was continued till column 1.
-For Control Row (Row H): 100 µL of DMSO was added in column 1- 10 and column 12. In column 10 and 11, 100 µL of 6.4 mM stock solution of trans cinnamaldehyde was added. Serial dilutions were prepared by transferring 100 µL from column 10 to column 9 (till column 7). Column 1-6 served as negative control (DMSO), column 7-11 served as positive control and column 12 served as no cell blank

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
After 24 ± 2 hours of incubation, medium from all the 4 plates (for single test item) was aspirated and discarded. It was replaced with 150 µL of DMEM containing 1% FBS without geneticin. The 25 fold dilution of the 100X master plate was performed into a fresh plate (10 µL test solution + 240 µL of DMEM with 1% FBS without geneticin). Resulting 4X plate was further distributed to assay plates: 50 µL of these stock solutions were added 3 white assay plates and one cytotoxicity plate already containing 150 µL of culture medium. Plates were then covered with foil or petri-seal and incubated for 46 hours in 5 ± 1 % CO2 at 37 ± 1ºC.

CONCENTRATIONS TESTED
-Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.20 µg/mL to 400 µg/mL.
-Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 µM.


REPLICATES: The study was composed of 3 independent repetitions. For each repetition the test item and the controls were replicated on three independent plates for the measurement of induction and one plate for the measurement of cytotoxicity. Two valid repetitions (experiment 1 and 3) were considered for final evaluation The reason for conducting 3 experiments was the failure of negative control (i.e. variation in the solvent control well was high) to meet the acceptance criteria (experiment 2).

LUCIFERASE ACTIVITY.
-Procedure: After 48 ± 2 hours of incubation, the medium from 96 well white assay plates was aspirated and discarded. Cells were washed once with 150 µL of DPBS. After washing, 20 µL of passive lysis buffer was added in each well. Cells were then incubated for 30 ± 10 minutes at 37 °C. After incubation, the plates with cell lysate were placed in luminometer. 50 µL of 1X Luciferase substrate was added and luciferase activity was recorded for 2 seconds.

CITOTOXICITY ASSESSMENT.
-Procedure: Master mix was prepared by combining 27 µL of MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin. After 48 ± 2 hours of incubation, the medium from 96 well transparent plate was replaced with 227 µL above prepared master mix. The plate was covered with foil and incubated for 4 hours. After incubation, the medium was removed and 200 μL of a 10% SDS solution was added to each well. The plate was sealed and covered with a foil and placed in the incubator for overnight incubation. Following incubation the absorption at 570 nm was measured.

DEVIATIONS FROM OECD GUIDELINE: No.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The gene induction for positive control was found to be >1.5 at concentrations of 32 µM and 64 µM in both the repetitions. The EC1.5 value for positive control was found to be 36.37 µM and 26.21 µM in experiment 1 and 3, respectively. The average gene induction for positive control at 64 µM was found to be 1.88 and 7.30 for experiment 1 and 3, respectively. Clear dose response with increasing gene induction at increasing dose was observed for trans cinnamaldehyde in experiment 1 and experiment 3.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

53.49 %

At concentration:

25 other: µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: induction units

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

6.24 %

At concentration:

12.5 other: µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: induction units

Key result

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

5.45 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(EC1.5 < IC30)

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

4.24 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(EC1.5 < IC30)

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

10.28 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(EC1.5 < IC30)

Key result

Run / experiment:

run/experiment 3

Parameter:

IC30 [442D]

Value:

11.35 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(EC1.5 < IC30)

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: yes, the 10 proficiency substances recommended in the OECD guideline 442D were performed, obtaining values that fall within the respective reference range for 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition 1 was 18.25% and for repetition 3 was 18.83% which are less than 20%.
Variability in the experiment 3 was in fact 24.29 %. Grubbs' test was applied and since the deviation was higher due to a single outlier in the 18 wells, results are considered valid. After removal of outlier variation in the negative control was 18.83%

- Acceptance criteria met for positive control: The luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The EC1.5 value for positive control was found to be 36.15 µM and 13.27 µM in experiment 1 and 3, respectively. The average gene induction for positive control at 64 µM was found to be 2.13 and 8.79 for experiment 1 and 3, respectively. The latter criterion is not fulfilled, however, clear dose response with increasing gene induction at increasing dose was observed for trans cinnamaldehyde in experiment 1 and experiment 3. Thus, the acceptance criteria are met.

Table 1: Mean Summary of Imax, EC1.5, IC30 and IC50 values of test item 

I_max

Test Item	Rep1	Rep3	Average
N-Ǹ̀-(Methylenedi-P-Phenylene)Bis(Aziridine-1-Carboxamide) (Poly-U-01)	53.49*	6.24*	29.86

 

EC_1.5

Test Item	Rep1	Rep3	GeoMean
N-Ǹ̀-(Methylenedi-P-Phenylene)Bis(Aziridine-1-Carboxamide) (Poly-U-01)	5.45µg/mL	4.24µg/mL	4.85µg/mL

 

IC₅₀

Test Item	Rep1	Rep3	GeoMean
N-Ǹ̀-(Methylenedi-P-Phenylene)Bis(Aziridine-1-Carboxamide) (Poly-U-01)	16.30µg/mL	16.71µg/mL	16.50µg/mL

 

IC₃₀

Test Item	Rep1	Rep3	GeoMean
N-Ǹ̀-(Methylenedi-P-Phenylene)Bis(Aziridine-1-Carboxamide) (Poly-U-01)	10.28µg/mL	11.35µg/mL	10.80µg/mL

Table 2: Mean Summary of Imax, EC1.5, IC30 and IC50 values of Positive control

Trans-Cinnamaldehyde	Imax	EC1.5	IC50	IC30
Rep 1	2.13	36.15	-	-
Rep 3	8.79	13.27	-	-

Table 3: Summary of Mean Induction of the test item (Two Experiments)

Concentration (µg/mL)	N-Ǹ̀-(Methylenedi-P-Phenylene)Bis(Aziridine-1-Carboxamide) (Poly-U-01)
	Mean	SD
0.20	0.95	0.21
0.39	0.95	0.13
0.78	1.03	0.22
1.56	0.96	0.21
3.13	1.03	0.14
6.25	1.93	0.33
12.5	5.36	1.25
25	27.05	37.39
50	0.01	0.02
100	0.01	0
200	0.04	0
400	0.04	0

Interpretation of results:

other: KeratinoSensTM test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442D.

Conclusions:

Under the experimental conditions the test substance may be classified as skin sensitizer using the KeratinoSensTM test method.

Executive summary:

The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.20 µg/mL to 400 µg/mL, positive control trans cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control DMSO were placed in the seeded plates and incubated for 48 hours ± 2 hour at 37ºC, 5% CO2. The study was composed of 3 independent repetitions and out of these, two valid repetitions (experiment 1 and 3) were considered for final evaluation. The reason for conducting 3 experiments was the failure of negative controls to meet the acceptance criteria (experiment 2). All validity criteria were fulfilled. For the test item, calculated Imax values were higher than 1.5 and EC1.5 values were lower than 200 µg/mL in the 2 repetitions. Also at EC1.5 concentration the reduction of viability was determined lower than 30% (i.e. EC1.5 < IC30). Finally, the observed dose response for luciferase induction was also statistically significant. Thus a positive result can be predicted for skin sensitization using the KeratinoSensTM test method.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 June 2018 – 21 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (Annex I: In vitro skin sensitisation: human cell line activation test (h- CLAT))

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
- Solubility and stability of the test substance in the solvent/vehicle: The test item was checked for its solubility, in a suitable solvent before performing the DRF assay. Solubility of the test item was evaluated by following procedure:
1. Saline was used as a first solvent choice at a final concentration of 100 mg/mL. Considering purity of the test item as 99.4%, to achieve a concentration of 100 mg/mL, 100.61 mg of the test item was weighed and volume was made up to 1 mL with saline. The test item was found to be insoluble in saline, hence solubility in second solvent, i.e., DMSO was also evaluated.
2. DMSO was used as a second solvent choice at a final concentration of 500 mg/mL. Considering purity of the test item as 99.4%, to achieve a concentration of 500 mg/mL, 503.02 mg of the test item was weighed and volume was made up to 1 mL with DMSO. The test item was found to form suspension in DMSO, after 10 min of sonication. Based on the solubility results, DMSO was selected as a vehicle for the study.

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Details on study design:
TEST SYSTEM
-Cell line: THP-1 cell line procured from American Type Culture Collection (ATCC) (Batch nº 63176297)
-Culture: cells were cultured at 37ºC, 5% CO2 in RPMI-1640 (Gibco, Lot #1897264, 1897375) with 2 mM L-glutamine and 25 mM HEPES (according to h-CLAT DB-ALM protocol No 158) supplemented with 10% v/v FBS (Fetal Bovine Serum, Gibco, Lot#1841109, 1907413), 0.05 mM 2-Mercaptoethanol (Gibco, Lot #1852695) and appropriate antibiotics: 100 U/mL penicillin and 100 μg/mL streptomycin
(Gibco, Lot #1924795).
-Passage number: cells were used at passage 15 (DRF experiment-1), 16 (DRF experiment-2), 18 (DRF experiment-3), 22 (Expression study, experiment - 1), 23 (Expression study, experiment -2) and 24 (Expression study, experiment-3).
-Reactivity check: Yes, performed ca. 1 month after thawing with cells of passage 14 on positive controls DNCB and nickel sulphate, and negative control lactic acid.

CONTROLS
-Positive control: 2, 4- Dinitrochlorobenzene (CAS 97-00-7; Sigma Aldrich batch no STBD7009V; purity 99.9%)
-Medium Control: complete medium
-Solvent Control: DMSO diluted to 0.4% in complete medium, which was tested at a final concentration of 0.2 % in the plate.

PREPARATION OF CELLS
-Pre-culture: Before assay, THP-1 cells were pre-cultured in culture flasks for around 48 h when seeded at a density of 0.2 × 10^6 cells/mL. Cells were maintained in suspension at densities from 0.1 to 0.8 × 10^6 cells/mL.
-cells suspension: Pre-cultured cells were collected by centrifugation (130 x g, 4 oC, 5 min) and resuspended in fresh culture medium at a density of 2 × 10^6 cells/mL.
-Nº of culture plates: For dose range finding (DRF) assay, cells were distributed into a 96-well flat-bottom plate with 80 μL. For CD86/CD54 expression measurement, cells were distributed into a 24 well flat-bottom plate with 500 μL.
-Cell density: 1.6 × 10^5 cells/well (DRF assay) and 1 × 10^6 cells/well (CD86/CD54 expression measurement).

DOSE FINDING ASSAY
-Preparation of test item: Stock solution of concentration 500 mg/mL of the test item was first prepared. Further ten stock solutions were prepared by 2-fold serial dilutions using DMSO (0.98–500 mg/mL). These stock solutions were further diluted 250-fold into culture medium (to obtain working solutions).
-Preparation of negative control: Along with the medium control, the solvent control used was DMSO, tested at a final concentration of 0.2% in the plate.
-Application of test item and control: The working solutions of test item and control were mixed in a 1:1 (v/v) ratio with the cell suspensions prepared in the 96-well flat-bottom plate (final range of concentrations of test item in the plate was 1.95–1000 μg/mL). The treated plates were incubated for 23.5 hours at 37 ± 1ºC under 5 ± 1% CO2.
-cell staining: After 23.5 hours of exposure, cells were transferred into 96-well V-bottom plate and collected by centrifugation (250 x g, 4ºC, 5 min). The supernatants were discarded and the remaining cells were resuspended with 200 μL of staining buffer (phosphate buffered saline (Sigma, Lot #SLBT3740) containing 0.1% bovine serum albumin (HiMedia, Lot #0000268629)). Cells were washed twice with 200 μL of staining buffer. Finally, cells were resuspended in 200 μL of Propidium iodide (PI, Sigma, Lot #SLBM6232V)-staining buffer (final concentration of PI = 0.625 μg/mL).
-Cytotoxicity measurement and estimation of CV75 value:
Apparatus: flow cytometer (FACSVerseTM), Becton Dickinson (BD)
CV75 value: calculated according to equation stated in OECD TG 442E.
-Number of experiments: 3 runs. A third repetition was conducted to confirm results of first two experiments. Based on the results of three experiments, CV75 value of test item was determined.

CD86/CD54 EXPRESSION MEASUREMENT
-Preparation of test item: The test item was first diluted to the concentration corresponding to 500-fold of the 1.2 × CV75, as determined in the dose range finding assay. Then, 1.2-fold serial dilutions were performed in DMSO to obtain the stock solutions (eight concentrations ranging from 500 × 1.2 × CV75 to 500 × 0.335 × CV75) to be tested. These stock solutions were further diluted 250-fold in culture medium (working solutions).
-Preparation of positive control: A 10 mg/mL stock solution of DNCB was prepared in DMSO and diluted to 2 mg/mL. This was further diluted 250-fold in culture medium to obtain working solution of concentration 8 μg/mL. The working solution was used for treatment with the cell suspension in 1:1 ratio (final range of concentration in the plate was 4.0 μg/mL).
-Preparation of negative control: Along with the medium control, the solvent control used was DMSO, tested at a final concentration of 0.2% in the plate.
-Application of test item and controls: The working solutions of test item and controls prepared (500 μL) were mixed with 500 μL of suspended cells (1 x 10^6 cells) in a 1:1 ratio in a 24-well plate (final range of concentrations of test item in the plate was 5.2–18.6 μg/mL) and incubated for 23 hours and 30 minutes at 37± 1ºC under 5± 1% CO2.
-cell staining: After 23 hours and 30 minutes of exposure, cells were transferred from 24-well plates into sample tubes, collected by centrifugation (250 x g, 4ºC, 5 min) and then washed twice with 1 mL of staining buffer. After washing, cells were blocked with 600 μL of blocking solution (staining buffer containing 0.01% (w/v) globulin (Cohn Fraction II, III Human, lyophilized powder, Sigma, Lot# 017K7650V)) and incubated for 15 minutes on ice. After blocking, cells were split in three aliquots of 180 μL into a 96-well V-bottom plate.
The three groups of cells were centrifuged and the cell pellets were stained with 50 μL of FITC-labelled anti-CD86 (BD Pharmingen, Lot #6348610, 7090720), anti-CD54 (Dako, Lot #20044016) or mouse IgG1 (isotype) antibodies (Dako, Lot #20046409) for 30 minutes on ice. The antibodies were used by diluting 6:50 (v/v, for CD86) or 3:50 (v/v, for CD54) and IgG1 with staining buffer. After incubation, the cells were centrifuged (250 x g, 4ºC, 5 min) and were washed with 200 μL of staining buffer three times. Finally, cells were resuspended in 200 μL of PI-staining buffer (final concentration of PI = 0.625 μg/mL).
- CD86/CD54 measurement and cell viability:
Apparatus: flow cytometer (FACSVerseTM), Becton Dickinson (BD)
CD86/CD54 expression: Relative Fluorescence Intensity (RFI) was used as an indicator of CD86 and CD54 expression calculated according to equation stated in OECD TG 442E.
EC150/EC200 values: calculated according to equation stated in OECD TG 442E.
-Number of experiments: 3 runs. For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs for CD86/CD54 expression measurement were performed.

DEVIATIONS FROM OECD GUIDELINE: No.

Vehicle / solvent control:

DMSO

Negative control:

other: culture medium

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

% cell viability: ≥ 50% (all experiments)
% RFI CD86: ≥ 150% (all experiments)
% RFI CD54: ≥ 200% (all experiments)
See results on “Any other information on results incl. tables”

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Value:

15.5 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

5.1 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

63 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC150, CD86 [442E]

Value:

6.8 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC150, CD86 [442E]

Value:

6.8 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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: yes, the 10 proficiency substances recommended in the OECD guideline 442E were performed, obtaining values that fall within the respective reference range for 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative controls: yes, the cell viabilities of medium and solvent controls were found to be >90 % in all three experiments. In the solvent control, % RFI CD86 and % RFI CD54 were found to be ≤ 150% and ≤ 200% respectively in all three experiments. The MFI ratio of medium and solvent controls for both CD86 and CD54 to isotype control were found to be >105% in all three experiments.
- Acceptance criteria met for positive control: Yes, the RFI values of DNCB were found to be 274.80%, 335.79% and 401.31% for CD86 marker and 506.46%, 264.49% and 474.19% for CD54 marker, which met the assay acceptance criteria (RFI CD86 ≥ 150%, RFI CD54 ≥ 200%). The cell viability of positive control was found to be >70 % in all three experiments.
- Acceptance criteria met for test item: yes, the cell viability of test item was found to be >50% in 7/8 concentrations in all three experiments.

Table 1: Results of CV75, EC150 and EC200

Sr. N^o	Parameters	Expt.1	Expt.2	Expt.3	Average values (in µg/mL)
1	CV75 (in µg/mL)	16.4	10.0	20.0	15.5
2	EC150 (in µg/mL)	5.1	63	6.8	6.8*
3	EC200 (in µg/mL)	-	-	-	Negative

* = Median EC value of the three experiments

Table 2: Cell Viability, MFI and RFI Values of Positive Control in Expression Study

Expt. N^o	IgG1			% Cell Viability		MFI			% RFI CD86		% RFI CD54
	Total Count	THP-1 cells | Count (Number of Gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)	Observed values	Expected values	IgG1	CD86	CD54	Observed values	Expected values	Observed values	Expected values
1	31996	16580	12289	74.1	≥ 50	76.8	248	210	274.80	≥ 150	506.46	≥ 200
2	20375	12696	10531	82.9		87.5	247	229	335.79		264.49
3	25775	14616	12244	83.8		98.5	558	319	401.31		474.19

Table 3: Cell Viability, MFI and RFI Values of Medium Control in Expression Study

Expt. N^o	IgG1			% Cell Viability		MFI			Observed MFI ratio (in %)		Expected MFI ratio of CD86/IgG1 and CD54/IgG1
	Total Count	THP-1 cells | Count (Number of Gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)	Observed values (in %)	Expected values (in %)	IgG1	CD86	CD54	CD86/ IgG1	CD54/ IgG1
1	15155	12240	11284	92.2	>90	89.2	152	116	170.40	130.04	> 105%
2	13177	11105	10479	94.4		94.6	160	132	169.13	139.53
3	14654	11913	11265	94.6		94.1	188	137	199.79	145.59

Table 4: Cell Viability, MFI and RFI Values of DMSO Control in Expression Study

Expt. N^o	Total Count	THP-1 cells | Count (Number of gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)	% Cell Viability		MFI			Observed MFI ratio (in %)		Expected MFI ratio of CD86/IgG1 and CD54/IgG1	% RFI CD86		% RFI CD54
												Observed values	Expected values	Observed values	Expected values
				Observed values	Expected values	IgG1	CD86	CD54	CD86/ IgG1	CD54/ IgG1
1	14579	11905	11196	94.0	>90	87.7	150	114	171.04	129.99	>105%	99.20	<150	98.13	<200
2	13826	11371	10459	92.0		93.5	141	147	150.80	157.22		72.63		143.05
3	14732	12033	11364	94.4		93.5	208	140	222.46	149.73		121.94		108.39

Table 5: Cell Viability, MFI and RFI Values of Test Item in CD86/CD54 Expression Measurement

Expt.1
Conc (µg/mL)	IgG1			% Cell Viability	MFI			% RFI
	Total Count	THP-1 cells | Count (Number of gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)		IgG1	CD86	CD54	CD86	CD54
18.6	41267	26919	11144	41.4	67.9	134	97.4	106.10	112.17
15.5	32342	21516	11055	51.4	71.3	149	104	124.72	124.33
12.9	28808	19531	11113	56.9	73.5	161	107	140.45	127.38
10.8	27877	18412	11293	61.3	73.9	160	107	138.20	125.86
9.0	24379	16714	11369	68.0	75.3	167	109	147.19	128.14
7.5	23066	16008	11238	70.2	76.8	152	112	120.71	133.84
6.2	20895	14730	11373	77.2	79.3	182	121	164.85	158.56
5.2	19928	14129	11413	80.8	80.6	175	113	151.52	123.19

Expt.2
Conc (µg/mL)	IgG1			% Cell Viability	MFI			% RFI
	Total Count	THP-1 cells | Count (Number of gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)		IgG1	CD86	CD54	CD86	CD54
18.6	35287	22115	10256	46.4	80.2	148	153	142.74	136.07
15.5	26514	17414	10301	59.2	81.3	177	138	201.47	105.98
12.9	23037	15732	10328	65.6	81.1	209	150	269.26	128.79
10.8	21854	15081	10365	68.7	90	208	147	248.42	106.54
9.0	19958	14045	10357	73.7	85.5	212	149	266.32	118.69
7.5	18712	13344	10369	77.7	88.7	200	138	234.32	92.15
6.2	17638	12813	10370	80.9	88.2	205	145	245.89	106.17
5.2	16046	12181	10398	85.4	91.8	210	147	248.84	103.18

Expt.3
Conc (µg/mL)	IgG1			% Cell Viability	MFI			% RFI
	Total Count	THP-1 cells | Count (Number of gated cells)	THP-1 cells/ Propidium Iodide-A- | Count (Number of Live cells)		IgG1	CD86	CD54	CD86	CD54
18.6	35375	22893	11261	49.2	93.2	196	158	89.78	139.35
15.5	26957	18218	11236	61.7	94.1	230	177	118.69	178.28
12.9	25215	17030	11191	65.7	94.6	246	161	132.23	142.80
10.8	23089	15974	11269	70.5	96.4	250	165	134.15	147.53
9.0	21791	15353	11339	73.9	96.7	271	156	152.23	127.53
7.5	21466	14967	11342	75.8	98.5	281	156	159.39	123.66
6.2	19554	13965	11332	81.1	98.1	260	154	141.40	120.22
5.2	18051	13146	11393	86.7	97.6	268	152	148.82	116.99

Interpretation of results:

other: h-CLAT test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442E.

Conclusions:

Under the experimental conditions the test item may be classified as skin sensitizer using the in vitro h-CLAT test method.

Executive summary:

The h-CLAT test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442E, under GLP conditions. After solubility test, DMSO was selected as solvent. Concurrent negative controls, one consisting of solvent and other consisting of complete medium alone without test item were included in each assay. 2, 4- Dinitrochlorobenzene (DNCB) was used as a positive control at a single concentration of 4.0 μg/mL in DMSO. Three independent Dose Range Finding (DRF) experiments were performed at a final concentration range in the plate of 1.95–1000 μg/mL. The CV75 value of test item was found to be 15.5 μg/mL. Based on this value, three independent experiments of CD86/CD54 expression measurement (expression study) were performed at eight concentrations ranging from 5.2 to 18.6 μg/mL. Negative and positive controls met the specified acceptance criteria for the controls.Furthermore, the cell viability of test item was found to be >50% in 7/8 concentrations in all three experiments. In all experiments, a positive response was obtained for CD86 marker only, hence the prediction was “P1 +P1 +P1”. Based on these results, the test item was concluded as sensitizer in hCLAT assay. The median EC150 value of test item was found to be 6.8 μg/mL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

First key event (molecular initiating event):Weight of evidence. A DPRA skin sensitization test was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item was prepared at 100 mM in Milli-Q water and Cinnamaldehyde (positive control) was prepared at 100 mM in acetonitrile. Reference controls A, B and C were prepared with acetonitrile and Milli-Q water (control C) in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C±2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested in triplicate for both peptides. All validity criteria were fulfilled. Percent peptide depletion values of the test substance for Cysteine and Lysine were -122% (which shows significant co-elution) and 1%, respectively. Mean percent depletion was not calculated since co-elution was observed with cysteine peptide. Thus, the test substance cannot be classified as either “sensitizer” or “non-sensitizer” and hence the result of DPRA test is considered as “inconclusive”.

Second key event (Activation of keratinocytes):Weight of evidence. The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.20 µg/mL to 400 µg/mL, positive control trans cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control DMSO were placed in the seeded plates and incubated for 48 hours ± 2 hour at 37ºC, 5% CO2. The study was composed of 3 independent repetitions and out of these, two valid repetitions (experiment 1 and 3) were considered for final evaluation. The reason for conducting 3 experiments was the failure of negative controls to meet the acceptance criteria (experiment 2). All validity criteria were fulfilled. For the test item, calculated Imax values were higher than 1.5 and EC1.5 values were lower than 200 µg/mL in the 2 repetitions. Also at EC1.5 concentration the reduction of viability was determined lower than 30% (i.e. EC1.5 < IC30). Finally, the observed dose response for luciferase induction was also statistically significant. Thus a positive result can be predicted for skin sensitization using the KeratinoSensTM test method.

Third key event (activation of dendritic cells):Weight of evidence. The h-CLAT test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442E, under GLP conditions. After solubility test, DMSO was selected as solvent. Concurrent negative controls, one consisting of solvent and other consisting of complete medium alone without test item were included in each assay. 2, 4- Dinitrochlorobenzene (DNCB) was used as a positive control at a single concentration of 4.0 μg/mL in DMSO. Three independent Dose Range Finding (DRF) experiments were performed at a final concentration range in the plate of 1.95–1000 μg/mL. The CV75 value of test item was found to be 15.5 μg/mL. Based on this value, three independent experiments of CD86/CD54 expression measurement (expression study) were performed at eight concentrations ranging from 5.2 to 18.6 μg/mL. Negative and positive controls met the specified acceptance criteria for the controls. Furthermore, the cell viability of test item was found to be >50% in 7/8 concentrations in all three experiments. In all experiments, a positive response was obtained for CD86 marker only, hence the prediction was “P1 +P1 +P1”. Based on these results, the test item was concluded as sensitizer in hCLAT assay. The median EC150 value of test item was found to be 6.8 μg/mL.

Skin sensitisation: Weight of evidence. Integrated testing strategy (ITS). Based on the OECD Guidance Document on the reporting of defined approaches to be used within integrated approaches to testing and assessment for skin sensitization ((ENV/JM/MONO(2016)29), the “2 out of 3-sens ITS” approach as described in its annex 1 has been selected to allow the classification of the substance. The combination of test methods used in this strategy covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E or the modified Myeloid U937 Skin Sensitisation Test (mMUSST)].

According to this approach two concordant results addressing two different key events (KEs) indicate the sensitising potential, i.e. two positive results indicate a sensitiser, two negative results indicate a non-sensitiser.

Thus, the test substance does need to be classified as skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is classified as skin sensitiser (Cat 1) according to CLP Regulation no. 1272/2008.