Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 24 May 2019 to 22 July. 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

2018

Deviations:

yes

Remarks:

see "Principles of method if other guideline"

Principles of method if other than guideline:

The following deviation from the study plan was observed:
In experiment II, the culture plate was incubated with PI for 10 minutes instead of 5 minutes at room temperature, as stated in the study plan. This deviation doesn´t have any effect on the result of the study, because the incubation time for PI is not prescribed. More important is, that all samples are incubated for the same time, and this was given. Furthermore, the experiment was repeated.

The following deviation from the guideline was observed:
The reactivity check (RC) for the THP-1 cells used in this study wasn´t performed 2 weeks, but 3 weeks after thawing. Anyway, the RC was performed at least 2 weeks after thawing, the cells didn’t have a higher passage than 30 and were no longer in culture than 2 months. Therefore, this deviation is uncritical and doesn’t have any influence on the results of this study.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

EC Number:

276-521-4

EC Name:

Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

Cas Number:

72245-24-0

Molecular formula:

not applicable

IUPAC Name:

Benzoic acid, 2-hydroxy-, reaction products with formaldehyde, coupled with diazotized 5-amino-8-[[4-[(4-nitro-2-sulfophenyl)amino]phenyl]azo]-2-naphthalenesulfonic acid disodium salt

Test material form:

solid: particulate/powder

In vitro test system

Details on the study design:

Non-GLP Pre-Tests
The solubility of the test item was determined in a non-GLP pre-test in RPMI 1640, and DMSO. The test item was soluble or formed a turbid but stable suspension in medium at the concentrations 100 mg/mL (sonication for 5 minutes), 200 mg/mL (not sonicated, but made a dilution out of the 300 mg/mL stock) and 300 mg/mL (sonication for 10 minutes). Whether the test item was solved or formed a stable suspension could not be stated, because of the deep colour of the solution/suspension. However, 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 ± 5 nm. No emission was detected between 500 - 700 nm. Therefore, it is assumed that the test item has no influence on the result of the assay due to autofluorescence.

TEST SYSTEM
The OECD 442E indicates that the human monocytic leukaemia cell line, THP-1 should be used for the h-CLAT. The cells were purchased by CLS (Eppelheim, Germany).
-Cell Cultures:
THP-1 cells are stored in liquid nitrogen in the cell bank of LAUS GmbH 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 were maintained at densities from 0.1 to 1.0 * 106 cells/mL. Prior to using them for testing, the cells were qualified by conducting a reactivity check for the pre-test cells of passage 17 were used. For the main experiments, cells of passage 20, 21, and 10 were used. After thawing, the 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:
21 (20 for experiment IV and V) days after thawing (see chapter 11, page 27), a reactivity check of the cells was performed. For that, the two positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity, test concentration: 4 μg/mL) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity, test concentration: 200 μg/mL) as well as the negative control, lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity, test concentration: 1000 μg/mL) were used. These substances as well as all additional information are given by the OECD 442E. The experimental procedure was identical to the experiments in this study.

DEMONSTRATION OF PROFICIENCY
Prior to routine use, the validity of the h-CLAT test at LAUS GmbH was demonstrated in a non-GLP proficiency study. 12 proficiency substances were selected to represent the range of responses for skin sensitisation hazards. The expected h-CLAT prediction as well as the reference range were correctly obtained for 10 substances. All values fell within the respective reference range (CV75, EC150 for CD86, EC200 for CD54). 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 / II and IV / V 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) independently harvested cells were used (cells came from the same passage and were collected from different culture flasks.)

PRE-TESTS
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 the test item were tested with an incubation time of 24 h in the pre-test: 3000 μg/mL, 1500 μg/mL, 750 μg/mL, 375 μg/mL, 187.5 μg/mL, 93.8 μg/mL, 46.9 μg/mL and 23.4 μg/mL.
For that 1.6 * 105 cells / well of a 96-well plate were exposed to each concentration of the test item for 24 h at 37.0 ± 1.0 °C and 5.0 ± 0.5 % CO2. During treatment the plate is closed with a sealing tape to avoid cross-contamination. Following treatment, the cells were transferred into sample tubes and centrifuged for 5 min at 250 * g. Afterwards the supernatant was removed and the pellet was suspended in 200 μL staining buffer before 200 μL were transferred into one well of a 96-well plate. After that, the cells were washed two times with staining buffer (centrifugation: 250 * g for 5 min). After that, 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 PI. 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, except for the test item concentrations, where the cell viability was too low , data were acquired over a time period of 30 seconds*1 after starting the analysis. The cell viability
was automatically calculated by the flow cytometer. Based on these results, the highest concentration for the main experiments was defined.

The performance of all experiments was identical.
Eight test item concentrations were tested in experiment I to III: 3000 μg/mL, 2500 μg/mL, 2083.3 μg/mL, 1736.1 μg/mL, 1446.8 μg/mL, 1205.6 μg/mL, 1004.7 μg/mL and 837.2 μg/mL
and in experiment IV and V: 279.1 μg/mL, 334.9 μg/mL, 401.9 μg/mL, 482.3 μg/mL, 578.7 μg/mL, 694.4 μg/mL, 833.3 μg/mL, 1000.0 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 cells were transferred into sample tubes, collected by centrifugation (250 * g for 5 min) and then washed twice with 1 mL staining buffer. After that, the cells were resuspended in 600 μL blocking solution and incubated on ice for 15 min. Then, 180 μL of
the cell suspension 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 diluted FITC-labelled antibodies (anti-CD86, anti-CD54 or mouse IgG1) 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 antibodies 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. When the cell viability was too low, data were acquired over a time period of thirty seconds*1 after starting the analysis.

PREPARATION
To avoid the performance of a second pre-test since the concentration range would almost be the same, a stock solution containing 300 mg/mL (± 10 %), instead of 100 mg/mL test item in RPMI 1640 was prepared and sonicated until the test item was totally solved for the pre-test as well as for the experiments I to III. Subsequent dilution will finally yield a maximum concentration of 3000 μg/mL during treatment. For experiment IV and V a stock solution containing 100 mg/mL (± 10 %) test item in RPMI 1640 was prepared and sonicated until the test item was totally solved.
The stock solution was used to prepare a geometric series of 7 dilutions (factor 2 for the pretest; factor 1.2 for the experiments). Afterwards all concentrations were further diluted (1:50 fold) in complete culture medium (working solutions). Another 1:2 dilution was achieved by adding 1 part of each test item concentration to 1 part of the cell suspension in the treatment plate. In the end, the total dilution factor is then 1:100. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

CONTROLS
In accordance to the OECD 442E the controls were tested at only one concentration. All stock solutions were prepared freshly on the day of treatment. The selected concentrations were given by the OECD 442E guideline
Medium Control As medium control, complete culture medium was used.
Solvent Control for the positive control: Dimethyl sulfoxide (DMSO)
Positive control: DNCB
Solvent Control for Test Item: RPIM 1640

EXPOSURE CONCENTRATIONS AND DOSE SELECTION
The lowest measurable test item concentration was 125 µg/mL in both pre-tests. A CV75 could not be determined, because the next higher test item concentration (250 µg/mL) was too cytotoxic; not enough viable cells were left to be measured by flow cytometry. Therefore, the lowest cytotoxic concentration of both pre-tests (250 µg/mL) was used as highest concentration for the experiments. A 100x stock solution of 25 mg/mL test item was prepared and used for a geometric series of 7 dilutions (factor 1.2). Afterwards all concentrations were further diluted (1:50 fold) in culture medium (working solutions). Another 1:2 dilution was achieved by adding 1 part of each test item concentration to 1 part of the cell suspension in the treatment plate. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

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 the Relative fluorescence intensity RFI:
RFI = (MFI(chemical-treated cells) - MFI(chemical-treated isotype control cells)) / (MFI(solvent-treated control cells) - MFI(solvent-treated isotype control cells)) *100
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".

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 isotype 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 these criteria are not completely fulfilled, the acceptability of the study should be evaluated by the study director. In case of an invalid experiment, the experiment will be repeated under the same conditions.
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 concentration of a test item, a negative result is acceptable even if the cell viability is above 90 %.

Results and discussion

Positive control results:

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.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: experiments I, II and III

Parameter:

other: RFI of CD86 (%)

Remarks:

at all conc. with a cell viability ≥ 50 %

Value:

100

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A calculation of the EC150 and/or EC200 was not possible since none of the RFI values was above 150 (for CD86) and /or 200 (for CD54) at any of the tested concentrations.

Results 2

Run / experiment:

other: experiments I, II and III

Parameter:

other: RFI of CD54 (%)

Remarks:

at all conc. with a cell viability ≥ 50 %

Value:

100

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A calculation of the EC150 and/or EC200 was not possible since none of the RFI values was above 150 (for CD86) and /or 200 (for CD54) at any of the tested concentrations.

Results 3

Run / experiment:

other: Experiment IV and V

Parameter:

other: RFI of CD86 and CD54 (%)

Remarks:

at all concentrations with cell viability > 50% and citotoxicity effects

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A calculation of the EC200 was not possible. No RFI value lower than 200 is available du to cytotoxicity.

Other effects / acceptance of results:

PRE-TEST
None of the controls induced a cytotoxic effect. Since the cell viabilities of medium and solvents controls were higher than 90 % the pre-test was valid. The highest concentration (3000 μg/mL) was cytotoxic, however the logCV75 could not be calculated due to a missing viability value for the highest concentration, which was too cytotoxic in the pre-test. So, the first cytotoxic concentration, which was the highest, was used for the experiments (3000 μg/mL, 2500 μg/mL, 2083.3 μg/mL, 1736.1 μg/mL, 1446.8 μg/mL, 1205.6 μg/mL, 1004.7 μg/mL and 837.2 μg/mL).

Substance Concentration Viability
[µg/ml] [%]
Medium - 99.31
Solvent Control Item - 98.8
Solvent control positive control - 99.05
Positive control 4 89.76
Test Item 23.4 99.17
Test Item 46.9 98.33
Test Item 93.8 97.13
Test Item 187.5 96.62
Test Item 375 96.95
Test Item 750 97.61
Test Item 1500 92.06
Test Item 3000 83.33


EXPERIMENT I
A calculation of the EC150 and/or EC200 was not possible since none of the RFI values was above 150 (for CD86) and /or 200 (for CD54) at any of the tested concentrations.
EPERIMENT II
A calculation of the EC150 and/or EC200 was not possible since none of the RFI values was above 150 (for CD86) and /or 200 (for CD54) at any of the tested concentrations.
EXPERIMENT III
A calculation of the EC150 and/or EC200 was not possible since none of the RFI values was above 150 (for CD86) and /or 200 (for CD54) at any of the tested concentrations.
EXPERIMENT IV
A calculation of the EC200 was not possible. No RFI value lower than 200 is available due to cytotoxicity.
EXPERIMENT V
A calculation of the EC200 was not possible. No RFI value lower than 200 is available du to cytotoxicity.


ACCEPTABILITY
All validity criteria were met. Therefore, the study is considered as valid.

PREDICTION MODEL
For CD86/CD54 expression measurement, each test item is tested in at least two inde-pendent 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. Then, the study is completed. If the first two runs are not concordant 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. 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

CLASSIFICATION
In accordance to 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 up-regulate the cell surface marker expression of THP-1 cells.

Applicant's summary and conclusion

Interpretation of results:

other: potential to activate dendritic cells

Conclusions:

THe test item is considered to have the potential to activate dendritic cells and therefore to up-regulate the cell surface marker expression of THP-1 cells.

Executive summary:

This in vitro study was performed to assess the potential of the test item to activate dendritic cells and therefore to up-regulate the cell surface marker expression of THP-1 cells 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.

In total one pre-test and five experiments (experiment I - V) with a treatment period of 24 hours were performed.

For the experiments I to III, the highest nominal applied concentration (3000 μg/mL) was chosen, based on the results obtained in the pre-test. Due to cytotoxicity and very low RFI values in experiment I and III, the highest nominal applied concentration (1000 μg/mL) was chosen for experiment IV and V.

A geometric series (factor 1.2) of 7 dilutions thereof 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 and were found to be suitable for the experiments.

All acceptance criteria were met and therefore, the study was considered valid. Cytotoxicity occurred in all experiments. In experiment II all analysable concentrations were below 49 % viability. In experiment I and III, the viability values were between 87 % - 36 %, but didn´t show any dose response, additionally the RFI values for all concentrations of the test item were extremely low compared to the solvent controls, a false negative result couldn´t be excluded.

Therefore, experiment IV and V were performed using 1000 μg/mL as highest test item concentration, followed by a clear dose response and positive RFI values for CD54 for all test item concentrations with cell viability ≥ 50 % for both experiments. The highest two test item concentrations in both experiments were cytotoxic (viability < 50 %), the generated values are not reliable. The values are not included in analysis. In conclusion, it can be stated that under the experimental conditions of this study, the test item, Acid Brown 362, was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and therefore to up-regulate the cell surface marker expression

of THP-1 cells.