Dimethylamine-borane (1:1)

Endpoint summary

Description of key information

In a skin sensitization study conducted according to OECD 442E with Dimethylamine-borane (Purity 99.33%) in 0.9% NaCl, the sensitization potential of the test item was assessed on the basis of the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). CD86 showed no upregulation in Experiment I, upregulation to 165% in Experiment II and 256% in Experiment III.

In a dermal sensitization study conducted according to OECD 442D with Dimethylamine-borane (Purity 99.33%) in DMSO, the sensitization potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™.

For both experiment I and II, Imax was greater than a 1.5-fold increase, cell viability was greater than 70% and the value for EC1.5 was less than 1000 µM.

According to these both studies, the test item is considered to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-18 to 2018-01-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E

Version / remarks:

Adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals. Moreover, this test method is able to detect chemicals that cause skin sensitization and allows hazard identification.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +5 °C; monitor temperature, store in tightly closed container in a cool, well-ventilated place

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was freshly prepared immediately prior to use. The test item was dissolved in 0.9% NaCl solution at a concentration of 100 mg/mL. Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 50 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent (0.9% NaCl solution) was present at a constant volume ratio of 1% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Details on the study design:

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

CELL LINE:
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (< 30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10^6 cells/mL.
Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/ 100 µg/mL streptomycin at 37 +/- 1 °C and 5% CO2.

PRE-EXPERIMENTS:
Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 µg/mL and nickel sulphate at a final concentration of 100 µg/mL served as positive control while lactic acid at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

EXPERIMENTAL PROCEDURE:
DOSE FINDING STUDY:
Starting from 100 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 50-fold into culture medium (working solutions). Since the test item was dissolved in 0.9% NaCl and the top concentration of 1000 µg/mL was non-toxic, the maximum concentration was re-determined by performing a new cytotoxicity test. Hereby, the highest possible stock concentration was 150 mg/mL in 0.9% NaCl. The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution.
For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 106 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well). The solvent controls and the working solutions of the test item were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2.
After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer. 200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL.The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated. The CV75 value, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.
CD54 and CD86 EXPRESSION:
The test item was dissolved using 0.9% NaCl as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.2²; CV75/1.2³; CV75/1.2^4; CV75/1.2^5; CV75/1.2^6. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 100-fold of the 1.2 × CV75. Then,1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 50-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor. For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of
0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min. All antibodies were diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. Moreover, the cell viability was calculated.

EVALUATION OF THE RESULTS:
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used. Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise, the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is < 90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities > 90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.
For test chemicals classified as sensitiser the effective concentration 150 for CD86 (EC150) and the effective concentration 200 for CD54 (EC200) can be calculated. For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs should be performed for CD86/CD54 expression measurement. The EC150 and EC200 values are the median value of the ECs calculated from three independent runs. In order to select the median value, three independent runs are necessary. If only two of three independent runs meet the positive criteria, the higher EC150 or EC200 of the two calculated values is adopted.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 and 200% for CD54 were clearly exceeded.

Run / experiment:

other: 1

Parameter:

other: CD86 upregulation

Value:

102

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86 upregulation

Value:

165

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 3

Parameter:

other: CD86 upregulation

Value:

256

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: CD54 upregulation

Value:

78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD54 upregulation

Value:

90

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 3

Parameter:

other: CD54 upregulation

Value:

103

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:

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

Results:

In the present study Dimethylamine-borane (DMAB) was dissolved in 0.9% NaCl. For the dose finding assays stock solutions with concentrations ranging from 100 mg/mL to 0.78 mg/mL for run 1 and with concentrations ranging from 150 mg/mL to 1.17 mg/mL for run 2 were prepared by a serial dilution of 1:2, respectively. Cells were incubated with the test item for 24 h at 37 °C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps: 1500, 1250, 1041.67, 868.06, 723.38, 602.82, 502.35 and 418.62 µg/mL. Cells were incubated with the test item for 24 h at 37 °C. After exposure, cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining. No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 92.1% (CD86), 91.2% (CD54) and 91.1% (isotype IgG1 control) in the first experiment, to 84.2% (CD86), 84.2% (CD54) and 83.7% (isotype IgG1 control) in the second experiment and to 86.1% (CD86), 85.7% (CD54) and 86.1% (isotype IgG1 control) in the third experiment. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

No upregulation of the cell surface marker CD86 was observed in the first experiment. In the second experiment, the expression of the cell surface marker CD86 was upregulated to 165%. The upregulation above the threshold of 150% was observed at a concentration of 1500 µg/mL. Due to the not concordant results a third experiment was performed to make the final prediction. In the third experiment the expression of the cell surface marker CD86 was upregulated to 256%. An upregulation above the threshold of 150% was observed starting from a concentration of 1041.67 µg/mL and higher and at a concentration of 723.38 µg/mL. Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item considered to be a skin sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (394% experiment 1; 269% experiment 2, 417% experiment 3) and 200% for CD54 (251% experiment 1; 247% experiment 2, 227% experiment 3) were clearly exceeded. The controls confirmed the validity of the study. The viability of the solvent control was > 90% (94.5-95.9% experiment 1; 96.6-97.0% experiment 2, 96.4-97.7% experiment 3). The number of tested test item concentrations with cell viability > 50% was ≥ 4 (8 experiments 1, 2 and 3). The RFI for CD86 and CD54 of cells treated with the solvent DMSO was ≤ 150% (82% experiment 1; 104% experiment 2, 111% experiment 3) and ≤ 200% (88% experiment 1; 89% experiment 2, 98% experiment 3). The MFI ratio of the medium control and isotype IgG1control was ≥ 105% for CD86 (303% experiment 1; 217% experiment 2, 325% experiment 3) and CD54 (212% experiment 1; 144% experiment 2, 181% experiment 3). The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105% for CD86 (266% experiment 1; 223% experiment 2, 351% experiment 3) and CD54 (198% experiment 1; 140% experiment 2, 180% experiment 3).

Table 1: CD54 and CD86 Expression at 1500 µg / mL

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Experiment I
Medium Control	-	94.8	94.5	94.7	2164	1513	714	1450	799	100	100	303	212
Solvent Control	0.20%	95.9	95.0	95.5	1916	1426	720	1196	706	82	88	266	198
DNCB	4.00	85.8	85.4	86.0	5381	2440	665	4716	1775	394	251	809	367
Dimethylamine-borane (DMAB)	1500	92.1	91.2	91.1	2183	1291	704	1479	587	102	73	310	183
Experiment II
Medium Control	-	96.8	97.0	96.6	1626	1081	749	877	332	100	100	217	144
Solvent Control	0.20%	96.7	96.8	96.6	1653	1036	741	912	295	104	89	223	140
DNCB	4.0	85.0	88.5	88.5	3229	1509	780	2449	729	269	247	414	193
Dimethylamine-borane (DMAB)	1500.00	84.2	84.2	83.7	2221	996	775	1446	221	165	67	287	129
Experiment III
Medium Control	-	97.7	97.0	96.4	1991	1109	612	1379	497	100	100	325	181
Solvent Control	0.20%	97.1	97.0	97.1	2139	1095	609	1530	486	111	98	351	180
DNCB	4.0	83.4	83.2	82.5	7085	1810	706	6379	1104	417	227	1004	256
Dimethylamine-borane (DMAB)	1500.0	86.1	85.7	86.1	4226	1087	694	3532	393	256	79	609	157

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item can be considered to be a skin sensitiser in accordance with UN GHS category 1.

Executive summary:

In a skin sensitization study conducted according to OECD 442E with dimethylamine-borane (Purity 99.33%) in 0 .9% NaCl, the sensitization potential of the test item was assessed on the basis of the activation of dendritic cells using the in vitro human cell line activation test (h-CLAT). Cells were incubated with the test item for 24 h at 37 °C and later checked for cell viability and expression of CD86 and CD54 cell surface markers.

Sensitization was scored by measuring cell viability and checking the expression of both cell surface markers. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. CD86 showed no upregulation in Experiment I, upregulation to 165% in Experiment II and 256% in Experiment III. Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item can be considered to be a skin sensitiser.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-18 to 2018-01-09

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)

Version / remarks:

adopted 04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. Therefore, the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: +5 °C; monitor temperature, store in tightly closed container in a cool, well-ventilated place

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity ≥ 99%; AppliChem; Lot No.: 0000978834). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. Based on the DMSO stock solution, serial dilutions were made using the solvent to obtain twelve master concentrations of the test item (0.098 to 200 mM). The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent. These 1:25 diluted test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Details on the study design:

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

CELL LINE:
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number < 25 (passage 12, experiment 1; passage 11, experiment 2) were used. Cells were cultured in 75 cm² culture flasks (Greiner) in maintenance medium at 37 +/- 1 °C and 5% CO2 in a humidified incubator. For test material exposure, cells were cultured in medium.

LUCIFERASE ASSAY SYSTEM:
The luciferase activity was determined using the following products purchased from Promega. All components were used according to the instructions of the manufacture manual. The kit (Promega, Cat. No.: E1501, Lot No.: 0000265318) consisted of the following components relevant for this study:
- 10 vials Luciferase Assay Substrate (lyophilized)
- 10 x 10 mL Luciferase Assay Buffer
If freshly prepared, Luciferase Assay Substrate was dissolved in Luciferase Assay Buffer. If thawed from -80 °C, Luciferase Assay Reagent was allowed to equilibrate to room temperature prior to use.
Luciferase Cell Culture Lysis 5x Reagent
The kit (Promega, Cat. No.: E1531, Lot No.: 0000245622) consisted of the following components relevant for this study:
- 30 mL Luciferase Cell Culture Lysis 5x Reagent
Prior to use lysis buffer was diluted 1:5 with dist. water (Sigma; Lot No.: RNBF7110)

DOSE GROUPS:
Negative Control: DMSO: 1% (v/v) in test item exposure medium
Positive Control: CA: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
Test Item: 12 concentrations of the test item: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.50 µM, 125.0 µM, 250.0 µM, 500.0 µM, 1000 µM, 2000 µM
Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run.

EXPERIMENTAL PROCEDURE:
The incubation was performed in 96-well plates.A cell suspension of 8 × 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1× 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 25 times diluted master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated 96-well plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

LUCIFERASE ACTIVITY:
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (Gibco Life Science; Lot No.: 1877596). Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

CELL VIABILITY:
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

DATA ANALYSIS:
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p< 0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells. The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.

PREDICTION MODEL:
The test item is considered positive in accordance with UN GHS “Category 1” for skin sensitisation if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p< 0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.

Positive control results:

- The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.24 in experiment 1; 2.99 in experiment 2).
- The calculated EC1.5 was between 7 and 34 µM (17.22 µM in experiment 1; 17.36 µM in experiment 2).

Key result

Run / experiment:

other: Mean of Experiment I and II at 2000 µM concentration of the test item

Parameter:

other: max luciferase activity (Imax) induction

Value:

4.77

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: Mean of Experiment I and II

Parameter:

other: calculated EC1.5 [µM]

Value:

186.43

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 1, at 2000 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

4.82

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: experiment II, at 2000 µM

Parameter:

other: max luciferase activity (Imax) induction

Value:

4.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 1

Parameter:

other: calculated EC1.5 [µM]

Value:

184.23

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment 2

Parameter:

other: calculated EC1.5 [µM]

Value:

188.64

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:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a
prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant
results a third independent run is performed.
- For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test
comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
- The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this
value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC1.5 determining concentration.

ACCEPTANCE OF RESULTS:
- Acceptance criteria: The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.
- Acceptance criteria met for positive control: Yes

Table 1 : Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.11	1.04	1.07	0.05
	8.00	1.33	1.18	1.25	0.10
	16.00	1.46	1.46	1.46	0.00
	32.00	2.03	1.98	2.01	0.04	*
	64.00	3.24	2.99	3.12	0.18	*
Test Item	0.98	0.98	0.93	0.96	0.04
	1.95	1.07	1.00	1.03	0.05
	3.91	1.04	1.02	1.03	0.01
	7.81	1.09	0.97	1.03	0.09
	15.63	1.07	1.01	1.04	0.04
	31.25	1.13	0.97	1.05	0.11
	62.50	1.11	1.19	1.15	0.06
	125.00	1.15	1.09	1.12	0.04
	250.00	1.89	1.89	1.89	0.00	*
	500.00	2.51	2.85	2.68	0.24	*
	1000.00	3.29	4.04	3.66	0.53	*
	2000.00	4.82	4.72	4.77	0.07	*

* = significant induction according to Student’s t-test, p<0.05

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.

Executive summary:

In a dermal sensitization study conducted according to OECD 442D with dimethylamine-borane (purity 99.33%) in DMSO, the sensitization potential of the test item was assessed on the basis of the activation of keratinocytes using the in vitro KeratinoSens™. Cells were incubated with the test item for 48 h at 37 °C and later checked for luciferase activity.

Sensitization was scored by measuring maximum luciferase activity induction (Imax), cell viability and EC1.5. For both experiment I and II, Imax was greater than a 1.5 fold increase, cell viability was greater than 70% and the value for EC1.5 was less than 1000 µM.

Therefore, in this study, the test item is considered to be a skin sensitizer under UN GHS “Category 1”.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In two in vitro skin sensitisation studies according to OECD 442D and 442E, Dimethylamine-borane was tested positive for skin sensitisation. Based on the results, a classification of Dimethylamine-borane as Skin sensitiser Category 1 is warranted.