Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guidelines 442E - Annex II (U937 Cell Line Activation Test USensTM Assay)
Version / remarks:
9 October 2017
Deviations:
no
GLP compliance:
yes
Type of study:
other: U937 Cell Line Activation Test (USENS TM) Assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch no. 160405
- Expiration date of the lot/batch: 31 January 2019
- Purity test date: not specified, only the purity was given in the study report.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature desiccated
- Stability under test conditions: Not indicated
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed. The test item was either dissolved or suspended in complete medium and DMSO to a final concentration of 50 mg/mL. The test item formed a clear solution in complete medium at 50 mg/mL. In DMSO the test item formed clear solution at 50 mg/mL.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
In the main experiments the test item was dissolved in complete medium at 0.4 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL and 200, 180, 140 and 100 µg/mL in experiment 1 and 2, respectively in the 96-well plate.
No precipitation was observed at the end of the incubation period in the 96-well plates.
Test item concentrations were used within 1 hours after preparation.


FORM AS APPLIED IN THE TEST (if different from that of starting material)
Diluted in DMSO

T

In vitro test system

Details on study design:
Skin sensitisation (In vitro test system)
- Details on study design:
TEST SYSTEM:
-Model used: U937 human monocytes (Inducible CD86-expressing cells) from ATCC (American Type Culture Collection)

-Stockage: Stock cultures of these cells are stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test.

-Cell culture medium: Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).

-Conditions: All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 70 – 100 %) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 – 36.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

EXPERIMENTAL DESIGN:

-Plating of cells: Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 10E5 viable cells/mL. If the cell viability is < 90% the cells were not used. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.

-Number of experiments: Two experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated. In total 2 valid experiments were performed.

-Positive Control
2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared in RPMI. This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 µg/mL).

-Negative Control
Lactic Acid (LA, RS471) is used as negative control. On the treatment day, a solution at
10 mg/mL was prepared in RPMI medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 µg/mL).

-Treatment and doses: Cells are treated for 45 ± 3 hours with the selected doses. The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL. A untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL. In the second experiment cells were treated with four selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 100, 140, 180 and 200 µg/mL. After 45 ± 3 hours of exposure, wells were checked for precipitate.

- Antibodies: FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

FLOW CYTOMETRY METHOD
-Acquisition: Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
-Analysis: All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot.
The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity were analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
-Color Interferences: On IgG1 analysis: There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

ACCEPTABILITY CRITERIA
• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells is > 90%
• When DMSO is used as a vehicle, the validity of the DMSO vehicle control is assessed by calculating a DMSO S.I. compared to untreated cells, and the mean viability of the triplicate cells is > 90%. The DMSO vehicle control is valid if the mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells.
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
• No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected %CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
• The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
• Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%).

CALCULATIONS:
For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well.
The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) was calculated using the following formula:

CV70=C1+[((V1-70)/(V1-V2)x(C2-C1)+C1

Where:
V1 = the first percentage of viability above 70%
V2= the first percentage of viability below 70%
C1 = dose level corresponding to V1
C2 = dose level corresponding to V2

For each CD86 well culture, the percentage of induced CD86+ cells is calculated as: [absolute %CD86+ — absolute%IgG1+]

A stimulation index (S.I.) is calculated for each dose level as follows: SI = [([%CD86+ - %IgG1+] in the treated culture)/(Mean [%CD86+ - %IgG1+] of the vehicle cultures)]x100

The viability for each dose level are the mean of the IgG1 well and CD86 well.
The theoretical concentration at which the chemical induces a S.I. of 150 (i.e., 50% of CD86+ cells over the vehicle control) was calculated using the following formula:
EC150 = C1 + [(150 – S.I. 1)/(S.I. 2 – S.I. 1)x(C2-C1)]
Where:
S.I.1 = the first percentage of CD86+ below 150%
S.I.2 = the first percentage of CD86+ above 150%
C1 = dose level corresponding to S.I. 1
C2 = dose level corresponding to S.I. 2

INTERPRETATION
• For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
• The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 Geo Mean S.I. ≥ 150%).
• In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
• An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N) (Figure 1). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
• An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P) (Figure 1). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
• There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4)



Results and discussion

Positive control results:
Experiment 1 : The positive control (TNBS) showed a S.I. ≥ 409% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 92% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2 : The positive control (TNBS) showed a S.I. ≥ 716% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 101% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Stimulation Index
Run / experiment:
Test Item - Experiment 1
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Stimulation Index
Run / experiment:
Test Item - Experiment 2
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (97% and 98% in experiment 1 and 2, respectively). The cells were in these experiments incubated with TMAO anhydrous in a concentration range of
1.0 – 200 µg/mL and 100 – 200 µg/mL in experiment 1 and 2, respectively. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

Experiment 1
• No precipitation was observed at the end of the incubation period in the 96-well plates.
• The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
• No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with TMAO anhydrous. No EC150 could be calculated and is considered to be higher than 200 µg/mL.
• The test item showed no colour interference.
• The positive control (TNBS) showed a S.I. ≥ 409% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 92% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 2
• No precipitation was observed at the end of the incubation period in the 96-well plates.
• The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
• No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with TMAO anhydrous. No EC150 could be calculated and is considered to be higher than 200 µg/mL
• The test item showed no colour interference.
• The positive control (TNBS) showed a S.I. ≥ 716% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 101% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Both tests passed the acceptance criteria:
• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100% in experiment 1 and 2).
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and
≤ 25% in both experiments.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of
≥ 0.6% and < 1.5% in both experiments.
• No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.
In both experiments the positive and negative control were considered valid and the positive control fell within the historical control data. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Table1          
Overview Stimulation index of CD86 and Cell Viability in
Experiment 1 and 2 of TMAO anhydrous

Test items

Dose
(µg/mL)

% Viability (Mean)*

CD86-IgG1 S.I.*

Colour Interference S.I.*

Experiment

Experiment

Experiment

1

2

1

2

1

2

TMAO anhydrous

 

 

 

 

 

 

 

1

99

-

51

-

103

-

 

10

99

-

92

-

107

-

 

20

99

-

106

-

104

-

 

50

100

-

31

-

105

-

 

100

100

100

89

94

104

101

 

140

-

100

-

120

-

103

 

180

-

100

-

120

-

104

 

200

99

100

95

131

107

103

-      Not Applicable

Table2          
Overview Stimulation index of CD86 and Cell Viability in Experiment 1 and 2 of the Positive, Negative and Vehicle Control

Controls

% Viability (Mean)*

CD86-IgG1 S.I.*

Experiment

Experiment

1

2

1

2

LA1

100

100

72

101

LA2

100

100

65

86

LA3

100

100

92

94

TNBS1

99

100

467

716

TNBS2

99

100

508

589

TNBS3

99

100

409

701

 

IgG1 value (%)

CD86 basal expression (%)

Experiment

Experiment

1

2

1

2

RPMI1

0.7

0.6

3.4

3.0

RPMI2

0.6

0.8

4.0

4.0

RPMI3

1.0

0.6

3.7

3.0

RPMI Mean Viability

 

100%

100%

RPMI Drift

 

-11%

-14%

LA Drift

 

35%

0%

 

*          Red values are either below 70% viability, above 150S.I..

 

 

Table3          
Overview EC150and CV70Values

 

EC150(µg/mL)

CV70(µg/mL)

Test item Experiment 1

NA

NA

Test item Experiment 2

NA

NA

                              NA = Not applicable

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of the study, the test item TMAO anhydrous did not showed cytotoxicity and did not induced CD86 activity at any of the test concentrations in both experiments. Hence, TMAO anhydrous is classified as negative in the USENS according to CLP criteria.
Executive summary:

The objective of this in vitro GLP-compliant study is to evaluate the ability of TMAO anhydrous to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

TMAO anhydrous was a white powder.  TMAO anhydrous was dissolved in complete medium at 0.4 mg/mL.  In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200 μg/mL).  In the second experiment, a more narrow dose-response analysis was performed up to 200 µg/mL. Test item, positive control and negative control were exposed to the cells during 45±3 hours. After the incubation with test item or controls, cells were treated with IgG1 CD86 specific antibody.  Two independent experiments were performed.

Both experiments passed the acceptance criteria:

•       At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100% in experiment 1 and 2).

•       The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments.

•       At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments.

•       No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

In both experiments the positive (TNBS) and negative (LA) control were considered valid.  Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

TMAO anhydrous showed no toxicity (No CV70 value) and no biologically relevant induction of the CD86 activity (No EC150 value) was measured at any of the test concentrations in both experiments.   TMAO anhydrous is classified as negative in the U-Sens™ assay since negative results (< 150% increase) were observed at all test concentrations with a cell viability of >70% compared to the vehicle control.

In conclusion, TMAO anhydrous is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the described experimental conditions