Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 August to 16 August, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation)

Version / remarks:

2018

Deviations:

yes

Remarks:

The reactivity check (RC) for THP-1 cells was performed 16 days after thawing instead of 2 weeks. This deviation is not thought to have any influence on results of this study.

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

EC Number:

947-350-9

Molecular formula:

N.A. (UVCB)

IUPAC Name:

Reaction products of N-methyldiethanolamine and Boric Acid (1:1.5)

Test material form:

liquid: viscous

In vitro test system

Details on the study design:

Skin sensitisation in vitro test system: human Cell Line Activation Test (h-CLAT)

PRE-TEST (non-GLP)
The solubility of the test item was determined in a non-GLP pre-test in medium (DMEM and RPMI 1640) and DMSO. The test item was sufficiently soluble in medium (DMEM was used for the non-GLP pre-test, but RPMI 1640 was used for the study, which is not critical as the media act the same way regarding solubility) up to the concentration 500 mg/mL. Therefore, RPMI 1640 was used as solvent in the test.
A possible autofluorescence of the test item was determined using a 2475 Multi-λ Fluorescence detector and an excitation wavelength of 488 nm ± 5 nm. No emission was detected between 520 nm and 580 nm. Therefore, the autofluorescence of the test item has no influence on the result of the assay.

PREPARATION
First, a stock solution (first pre-test: 100 mg/mL, second pre-test: 500 mg/mL and both experiments: 309.5 mg/mL of the test item) in RPMI 1640 was prepared and used to prepare a geometric series of solutions (factor 2 for pre-tests; factor 1.2 for main experiments). Afterwards, all concentrations were further diluted (1:50) in complete culture medium (working solutions). Another 1:2 dilution was achieved by adding 1 part of each test item concentration and 1 part of the cell suspension to the treatment plate. In the end, the total dilution factor was 1:100. The stock solutions as well as the dilutions were freshly prepared on the day of treatment.

MEDIUM CONTROL: Complete culture medium

SOLVENT CONTROL FOR TEST ITEM: RPMI 1640 (Biochrom AG); the dilution of the solvent was performed in the same way as for the test item

SOLVENT CONTROL FOR POSITIVE CONTROL: Dimethyl sulfoxide (DMSO) 99.5 % (Carl Roth); final concentration: 0.2 %; the dilution of the solvent was performed in the same way as for the test item

POSITIVE CONTROL: 2,4-dinitrochlorobenzene (DNCB) > 99 % (Sigma Aldrich); final concentration: 0.2 %; a stock solution of 2 mg/mL of DNCB in DMSO was prepared and further diluted (1:250 fold) in complete culture medium (working solution); the working solution was then diluted (1:2 fold) with the cell suspension to achieve a final test concentration of 4 μg/mL (0.2 %)

CELL CULTURES
THP-1 cells are stored in liquid nitrogen to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. The THP-1 cells are routinely seeded every 2-3 days at the density of 0.1 – 0.2 * 106 cells/mL. They are maintained at densities from 0.1 to 1.0 * 106 cells/mL. Prior to using them for testing, cells are qualified by conducting a reactivity check. For the pre-tests, cells of passage 14 and 15 were used. For the main experiments, cells of passage 17 were used. After thawing, cells were cultivated in RPMI 1640 complete culture medium in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

REACTIVITY CHECK
16 days after thawing, a reactivity check of the cells was performed: the two positive controls and the negative control were used. The experimental procedure was identical to the experiments in this study (below). The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore, the cells were found to be suitable for the experiments. For the pre-test as well as the experiments, only cells which have successfully passed the reactivity check were used.

CHEMICALS AND MEDIA
The purity of the chemicals which were used was either “analytical grade” or “for microbiological purposes”. Composition is stated as nominal composition, exact weights differed by max. ± 10 %. The given volumes are exemplary for the composition of the media/solutions.
- Culture base medium RPMI 1640 (Biochrom AG) serving as base for complete culture medium: Fetal calf Serum (FCS) (10 %); Penicillin/Streptomycin 10.000 U/mL / 10.000 μg/mL (100 U/mL / 100 μg/mL); 2-Mercaptoethanol 100 mM (0.05 mM); and Sodium pyruvate 100 mM (2 mM)
- Stock solution (100 mM) of 2-Mercaptoethanol in RPMI 1640 100 mM
- Fetal Calf Serum (FCS) Superior (Biochrom AG)
- PBS Dulbecco, ready to use (Biochrom AG) serving as base for staining buffer: Bovine serum albumin (BSA) 0.1 %
- Penicillin/Streptomycin, 10,000 U/mL / 10,000 μg/mL (Biochrom AG)
- Sodium pyruvate, 100 mM (Biochrom AG)
- Propidium iodide stock solution (1.25 mg/mL): Propidium iodide 25 mg and PBS ad 20 mL
- Propidium iodide working solution (12.5 μg/mL) in PBS: Propidium iodide stock solution (1.25 mg/mL) 10 μL and PBS 990 μL
- Stock solution for blocking solution (1 % (w/v)): Globulin 10 mg and PBS Dulbecco ad 1 mL
- Blocking solution (0.01 % (v/v)): Staining buffer 99 % and Stock solution for blocking solution 1 %
- Antibodies: (i) FITC (Fluorescein isothiocyanate)-anti-human-CD86, Clone: Fun-1, supplier BD-PharMingen; (ii) FITC-anti-human-CD54, Clone: 6.5B5, supplier DAKO; (iii) FITC mouse IgG1 (= Isotype control) supplier DAKO; and (iv) Globulin, Cohn fraction II, III, Human, supplier Sigma

TEST VESSELS
All vessels used were made of glass or sterilisable plastic. In case of non-sterilisation by the manufacturer, they were sterilised before usage in a heating chamber or autoclave. The test was performed on 96- and 24-well plates. For the transfer of the culture medium, pipettes were used. Glass measuring flasks and cylinders with conformity sign and standard laboratory material were also used.

DEMONSTRATION OF PROFICIENCY
Prior to routine use, the validity of the h-CLAT test was demonstrated in a proficiency study. In this study, 10 proficiency chemicals (indicated by the OECD 442E guideline) were tested. Fulfilling the requirements of the guideline, 8 of the results were correctly categorised. Therefore, the proficiency of the test system was demonstrated. For all control substances, historical data are available which demonstrate the reliability and the validity of those substances.
Prior to use in the pre-test and the experiments, the proficiency of the cells was demonstrated in a reactivity check. Only the cells which passed the reactivity check were used for the pre-test and the experiments. The runs for Experiment I and II were performed on the same day, provided that for each run: a) independent fresh stock solutions and working solutions of the test item and antibody solutions were prepared and b) inde-pendently harvested cells were used (cells came from the same passage and were collected from different culture flasks.)

PRELIMINARY CYTOTOXICITY TEST
- A preliminary cytotoxicity test was performed to determine the concentrations to be used for the CD86/CD54 expression measurement in the main experiments.
- The following 8 concentrations of test item were tested (incubation time of 24 h): 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 μg/mL.
- Performance: 1.6 * 105 cells / well were plated into a 96-well plate and exposed to each concentration of the test item for 24 h ± 0.5 h at 37.0 ± 1.0 °C and 5.0 ± 0.5 % CO2. Following treatment, the cells were transferred into sample tubes and centrifuged (250 * g for 5 min). The supernatants were discarded, the remaining cells were resuspended with 200 μL staining buffer, transferred into a 96-well plate and washed three times with staining buffer (centrifugation: 250 * g for 5 min). Finally, the cells were resuspended in 150 μL staining buffer and 7.5 μL PI solution (12.5 μg/mL) were added to achieve a final concentration of 0.595 μg/mL. The plate was incubated for 15 min on ice and 5 minutes at room temperature in the dark. Afterwards, the PI uptake was analysed by flow cytometry with the acquisition channel PerCP (corresponds to the FL-3 channel of the predecessor model). In total, 10,000 (± 10 %) viable cells (PI negative) were acquired. The cell viability was automatically calculated by the flow cytometer. For the evaluation of the CV75 value, a validated Microsoft Excel® spreadsheet was used. Based on these results, the concentrations for the main experiments were defined.
- Results: none of the controls induced a cytotoxic effect in the first pre-test. Since the cell viabilities of medium and solvents controls were higher than 90 % and the viability of all test item concentrations were > 50 %, the first pre-test was valid.
Since none of the tested concentrations induced a cytotoxic effect with RPMI 1640 as solvent and the test item was soluble at the concentration 500 mg/mL a second pre-test (pre-test 2) using the following concentrations was performed: 5000, 2500, 1250, 625, 312.5, 156.3, 78.1 and 39.1 μg/mL.
- None of the controls induced a cytotoxic effect in the second pre-test. Since the cell viabilities of medium and solvents controls were higher than 90 %, the second pre-test was valid.
- Since the highest test item concentration induced a cytotoxic effect, the CV75 was calculated: logCV75 is: 3.41, corresponding to a CV75 of 2579.22 μg/mL.
- On the basis of the CV75 vslue obtained, the maximum test item concentration for the experiments (1.2 * CV75) was calculated as: 3095 μg/mL

EXPOSURE CONCENTRATIONS AND DOSE SELECTION
Since a reduction of the viability was detected in the pre-test 2, the maximal test item concentration to be tested in the experiments corresponded to 1.2 * CV75 (calculated in the pre-test): 3095 μg/mL.
To achieve the highest test item concentration, a 100 x stock solution in RPMI 1640 was prepared and diluted.

PROCEDURE
The performance of experiment I and II was identical. Eight test item concentrations were tested in each experiment (863.8, 1036.5, 1243.8, 1492.6, 1791.1, 2149.3, 2579.2 and 3095.0 μg/mL). 1 * 106 cells / well of a 24-well plate were exposed to each concentration of the test item for 24 h ± 0.5 h at 37.0 ± 1.0 °C and 5.0 ± 0.5 % CO2.
During treatment, the plates were closed with a sealing tape to avoid cross-contamination. Following treatment, the solutions were transferred into sample tubes and washed twice with 1 mL staining buffer (centrifugation: 250 * g for 5 min). After that, the cells were resuspended in 600 μL blocking solution and incubated on ice for 15 min. Then, 180 μL of the cell sus-pension were added in three wells of a 96-well round bottom plate respectively. The plates were centrifuged at 250 * g for 5 minutes and the supernatant was discarded. After that, 50 μL of the three FITC-labelled antibody solutions were added in one of the three wells respectively and the plate was incubated on ice for 30 min in the dark. Before use, the anti-bodies were diluted 3:25 (v/v) for CD86, 3:50 (v/v), for CD54 and IgG1 with staining buffer.
After the incubation on ice, the cells were washed 3 times with 150 μL staining buffer (centrifugation: 250 * g for 5 min) before 150 μL staining buffer as well as 7.5 μL PI working solution were added to each well. Afterwards, the plate was incubated for 15 min on ice and 5 min at room temperature in the dark before measurement at the flow cytometer (BD FACS LyricTM). FITC-Detection: (Excitation: 488 nm / Detection filter 527/32; corresponding to the FL-1 channel). In total 10,000 viable cells (PI negative) were acquired and analysed.

DATA RECORDING AND ANALYSIS
The viability was directly measured and calculated by the flow cytometer and is given as % values. For the evaluation of the CD54/CD86 expression, the “mean fluorescence intensity (MFI)” was also analysed by the software of the flow cytometer. For calculation of the end results, a validated Microsoft Excel® spreadsheet file was used.

Calculation of CV75 in Pre-test: Log CV75 = ((75 - c) * Log (b) - (75 - a) * Log (d)) / (a - c)
where:
a = minimum value of cell viability over 75 %
b = concentration showing the value of cell viability of a
c = maximum value of viability below 75 %
d = concentration showing the value of cell viability of c

Calculation of the Relative fluorescence intensity RFI: RFI = (MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) * 100 / (MFI of solvent-treated control cells - MFI of solvent-treated isotype control cells)
RFI values of the solvent controls are calculated by using the same formula. However, "MFI of chemical" is replaced with "MFI of solvent", and "MFI of solvent" is replaced with "MFI of medium control".

Results and discussion

Positive control results:

Experiment I: CD86 = 88.47 % viability; RFI value = 515 %; CD54 = 88.73 % viability; RFI value = 256 %
Experiment II: CD86 = 87.36 % viability; RFI value = 547 %; CD54 = 86.56 % viability; RFI value = 267 %

In vitro / in chemico

Results

Results 1

Run / experiment:

other: I

Parameter:

other: RFI value of CD86

Remarks:

greater than 150 in all concentrations except highest two which demonstrated cytotoxicity

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Run / experiment:

other: I

Parameter:

other: RFI value of CD54

Remarks:

greater than 200 in 4 out of 8 concentrations tested

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Run / experiment:

other: II

Parameter:

other: RFI value of CD86

Remarks:

greater than 150 in all concentrations except highest two which demonstrated cytotoxicity

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 4

Run / experiment:

other: II

Parameter:

other: RFI value of CD54

Remarks:

greater than 200 at all concentrations except lowest two

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:

ACCEPTABILITY
The assay is considered acceptable if it meets the following criteria:
- The cell viabilities of medium and solvent/vehicle controls are higher than 90%.
- In the solvent/vehicle control, RFI values of both CD86 and CD54 do not exceed the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to iso-type control should be > 105%.
- In the positive control, RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 % and CD54 RFI ≥ 200 %) and cell viability should be more than 50 %.
- For the test item, the cell viability should be more than 50 % in at least four tested concentrations in each run.
If any of these criteria is not met, the experiment is considered not valid and needs to be repeated.
Negative results are acceptable only for test items exhibiting a cell viability of less than 90 % at the highest concentration tested. If the cell viability at 1.2 × CV75 is equal or above 90 % the negative result should be discarded. In such a case, another pre-test has to be performed to refine the dose selection. It should be noted that when 5000 μg/mL in PBS or medium, 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test con-centration of a test item, a negative result is acceptable even if the cell viability is above 90 %.
All validity criteria were met. Therefore, the study is considered valid.

CLASSIFICATION
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction. 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, otherwise the h-CLAT prediction is considered negative:
− The RFI of CD86 is ≥ than 150 % at any tested concentration with cell viability ≥ 50 %
− The RFI of CD54 is ≥ than 200 % at any tested concentration with cell viability ≥ 50 %
If the first two runs are concordant for both markers, a third experiment is not necessary and the study is completed. If the first two runs are not in accordance for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered negative. On the other hand, if the third run is positive for either marker or for both markers, the h-CLAT prediction is considered positive.
In case of a negative result, special care should be taken if the test item
− has a Log Kow > 3.5, as those results should not be considered, however, positive results obtained with those test chemicals can be used for analysis
− is a pro-hapten or a pre-hapten
− has an autofluorescence and is emitting at the same wavelength as FITC or as PI
In accordance with these classification criteria, the test item was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and therefore to be a potential skin sensitiser.

DISCUSSION
This in vitro study was performed to assess the sensitising potential of the test item by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. Two valid experiments with a treatment period of 24 hours were performed. In the experiments, the highest nominal applied concentration (3095 μg/mL) was chosen based on the results obtained in two pre-tests. A geometric series (factor 1.2) of 7 dilutions was prepared and tested. As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium. As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used Prior to the study, the cells used in the experiments were checked in a reactivity check test and were found to be suitable for the experiments. All acceptance criteria were met and therefore, the study was considered valid.

Since none of the tested concentrations induced a cytotoxic effect with RPMI 1640 as solvent and the test item was soluble at the concentration 500 mg/mL, a second pre-test (pre-test 2) using the following concentrations was performed: 5000, 2500, 1250, 625, 312.5, 156.3, 78.1 and 39.1 μg/mL. The highest test item concentration in pre-test 2 induced a cytotoxic effect, so the CV75 could be calculated and was 2579.22 μg/mL. On the basis of the CV75, the maximum test item concentration for the experiments (1.2 * CV75) was calculated to be 3095 μg/mL.

- Experiment I: RFI of CD86 was ≥ 150 % in the following concentrations: 863.8, 1036.5, 1243.8, 1492.6, 1791.1, 2149.3 μg/mL
- Experiment I: RFI of CD54 was ≥ 200 % in the following concentrations: 1492.6, 1791.1, 2149.3 and 3095.0 μg/mL
- Experiment II: RFI of CD86 was ≥ 150 % in the following concentrations: 863.8, 1036.5, 1243.8, 1492.6, 1791.1, 2149.3 μg/mL
- Experiment II: RFI of CD54 was ≥ 200 % in the following concentrations: 1243.8, 1492.6, 1791.1, 2149.3, 2579.2 and 3095.0 μg/mL

Therefore, in accordance with the classification criteria, the result of this study is positive. In conclusion, it can be stated that under the experimental conditions of this study, the test item was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and is a potential skin sensitiser.

Applicant's summary and conclusion

Interpretation of results:

other: activates a dentritic cell response

Conclusions:

The test item activates a dentritic cell response.

Executive summary:

In order to assess the sensitisation potential of the test item, an in vitro human Cell Line Activation Test (h-CLAT) was performed on the human monocytic leukaemia cell line (THP-1 cells) according to the OECD Guideline 442E (2018). In this assay, changes in the expression level of two cell surface markers, CD86 and CD54 (which are associated with activation of monocytes and dendritic cells), are quantified in two experiments in order to identify dendritic cell activation potential. The highest nominal concentration of 3095 µg/mL was chosen based on results of a preliminary test; a geometric series with factor 1.2 of 7 dilutions was prepared. A solvent control (RPMI 1640; 1 % in culture medium) and a positive control (2,4 -dinitrochlorobenzene (DNCB) were also tested in parallel to the test item. Cells were checked for reactivity prior to testing and were found to be suitable.

Since none of the tested concentrations induced a cytotoxic effect with RPMI 1640 as solvent and the test item was soluble at the concentration 500 mg/mL, a second pre-test was performed using a top concentration of 5000 μg/mL. As cytotoxicity was observed at the top concentration, the CV75 was able to be calculated and was found to be 2579.22 µg/mL. From this value, an appropriate maximum test item concentration value was calculated to be 3095 µg/mL.

- Experiments I and II: RFI of CD86 was ≥ 150 % in the following concentrations: 863.8, 1036.5, 1243.8, 1492.6, 1791.1, 2149.3 μg/mL

- Experiment I: RFI of CD54 was ≥ 200 % in the following concentrations: 1492.6, 1791.1, 2149.3 and 3095.0 μg/mL

- Experiment II: RFI of CD54 was ≥ 200 % in the following concentrations: 1243.8, 1492.6, 1791.1, 2149.3, 2579.2 and 3095.0 μg/mL

Since at least 2 out of 3 runs resulted positive, the test item is considered to have the potential to activate a dendritic cell response.