Registration Dossier

Administrative data

Description of key information

OECD TG 442C: positive in the DPRA and classified in the “low reactivity class”

OECD TG 442D: negative in the KeratinoSens assay

OECD TG 442E: negative in the U-Sens™ assay

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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
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
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)



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%).
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.

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

 

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

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes
Type of study:
direct peptide binding assay
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: no certificate of analysis was provided in the report but the purity was described at >98%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature dessicated
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in acetonitrile
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test item stock solution was prepared freshly.
For both the cysteine and lysine reactivity assay 12.37 mg of test item was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1647 µL ACN after vortex mixing and 1 minute of sonication to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively.
Any residual volumes were discarded.

FORM AS APPLIED IN THE TEST (if different from that of starting material) in solution in acetonitrile
Details on study design:
Skin sensitisation (In chemico test system)
- Details on study design:

Test system:
Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.

Source: JPT Peptide Technologies GmbH, Berlin, Germany.
Trifluoroacetic acid (TFA) >99%, Sigma Aldrich, Zwijndrecht, The Netherlands
EXPERIMENTAL DESIGN
-Preparation of Solutions for Cysteine Reactivity Assay:

Synthetic Peptide Containing Cysteine (SPCC) Stock Solution:
A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10 mg of SPCC in 19.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.

SPCC Reference Control Solutions :
Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.

A SPCC calibration curve was prepared as described in the table below (section "any other information of material and methods incl. tables")

-Preparation of Solutions for Lysine Reactivity Assay

Synthetic Peptide Containing Lysine (SPCL) Stock Solution:
A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 10 mg of SPCL in 19.31 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.

SPCL Reference Control Solutions:
Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.

SPCL Calibration Curve:
A SPCL peptide calibration curve was prepared as described in the table below(section "any other information of material and methods incl. tables")

-For cysteine and lysine, co-elution control, test item and positive control were prepared and details in tables below (section "any other information of material and methods incl. tables")

SAMPLE INCUBATIONS:

After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item samples) were placed in the autosampler in the dark and incubated at 25±2.5°C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25.5 hours and 24 hours, respectively. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours. Prior to HPLC PDA analysis the samples were visually inspected for precipitation.

-HPLC-PDA Analysis:
SPCC and SPCL peak areas in the samples were measured by HPLC PDA

The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test item. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer (according to OECD TG 442C)
Positive control results:
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 76.8% ± 1.5%. This was within the acceptance range of 60.8% to 100% with a SD that was below the maximum (SD <14.9%).

The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 54.1% ± 1.8%. This was within the acceptance range of 40.2% to 69.0% with a SD that was below the maximum (SD <11.6%).
Key result
Parameter:
other: SPCC depletion (%)
Run / experiment:
TMAO
Value:
20.4
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: SPCL depletion (%)
Run / experiment:
TMAO
Value:
0.2
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Mean of SPCC and SPCL depletion (%)
Run / experiment:
TMAO
Value:
10.3
Other effects / acceptance of results:
Solubility Assessment of the Test Item
At a concentration of 100 mM, TMAO anhydrous was soluble in ACN. Therefore this solvent was used to dissolve the test item in this DPRA study.

Cysteine Reactivity Assay
The correlation coefficient (r2) of the SPCC standard calibration curve was 0.994. Since the r2 was >0.99, the SPCC standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.521 ± 0.011 mM while the mean peptide concentration of Reference Controls C was 0.523 ± 0.028 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCC Depletion.

The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 5.8%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 19.29. The mean A220/A258 ratio ± 10% range was 17.36-21.22. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Lysine Reactivity Assay

The correlation coefficient (r2) of the SPCL standard calibration curve was 0.996. Since the r2 was >0.99, the SPCL standard calibration curve was accepted.

The mean peptide concentration of Reference Controls A was 0.511 ± 0.016 mM while the mean peptide concentration of Reference Controls C was 0.515 ± 0.031 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test item did not impact the Percent SPCL Depletion.

The CV of the peptide areas for the nine Reference Controls B and C was 3.7%. This was within the acceptance criteria (CV <15.0%) and confirms the stability of the HPLC run over time.

The mean area ratio (A220/A258) of the Reference Control samples was 15.26. The mean A220/A258 ratio ± 10% range was 13.73-16.78. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Table 7 : Acceptability of theDirect Peptide Reactivity Assay (DPRA)

 

Cysteine reactivity assay

Lysine reactivity assay

Acceptability criteria

Results for SPCC

Acceptability criteria

Results for SPCL

Correlation coefficient (r2) standard calibration curve

>0.99

0.994

>0.99

0.996

Mean peptide concentration RC-A samples (mM)

0.50 ± 0.05

0.521 ± 0.011

0.50 ± 0.05

0.511 ± 0.016

Mean peptide concentration RC-C samples (mM)

0.50 ± 0.05

0.523 ± 0.028

0.50 ± 0.05

0.515 ± 0.031

CV (%) for RC samples

B and C

<15.0

5.8

<15.0

3.7

Mean peptide depletion cinnamic aldehyde (%)

60.8-100

76.8

40.2-69.0

54.1

SD of peptide depletion cinnamic aldehyde (%)

<14.9

1.5

<11.6

1.8

SD of peptide depletion for the test item (%)

<14.9

3.3

<11.6

0.3

RC = Reference Control; CV = Coefficient of Variation; SD = Standard Deviation.

 

Table 8: SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification forthe Test Item

Test item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

DPRA prediction and reactivity classification

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

TMAO anhydrous

20.4%

±3.3%

0.2%

±0.3%

10.3%

Positive: Low reactivity

SD = Standard Deviation.

 

Interpretation of results:
other:
Conclusions:
Under the experimental condition of the study, in the cysteine reactivity assay the test item showed 20.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.2% SPCL depletion. The mean of the SPCC and SPCL depletion was 10.3% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

The objective of this in chemico GLP compliant study was to determine the reactivity of TMAO anhydrous towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OECD TG 442C method.

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm.  SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.  

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test item, were all within the acceptability criteria for the DPRA.

In the cysteine reactivity assay the test item showed 20.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.2% SPCL depletion.  The mean of the SPCC and SPCL depletion was 10.3% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.  TMAO anhydrous was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
February 2015
Deviations:
yes
Remarks:
No certificate of analysis was added in the report, only the purity of the test substance was detailed. Incubation time of cells with MTT was performed during 3 hours instead 4 required by the guideline.
GLP compliance:
yes
Type of study:
activation of keratinocytes
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

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 dissolved in DMSO to a final concentration of 200 mM (clear colourless)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM (clear)
- Preliminary purification step (if any): not specified
- Final dilution of a dissolved solid, stock liquid or gel: 200 mM
- Final preparation of a solid: not detailed

FORM AS APPLIED IN THE TEST (if different from that of starting material)
In solution in DMSO
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:

Test System
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

Basic medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).

Maintenance medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).

Exposure medium
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 65 – 100 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 – 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door.

Subculturing
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+4 in experiment 1 and P+6 in experiment 2.

Preparation of the Positive Control
The positive control used in the case of KeratinoSensTM is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted , so that the final concentration of the positive control ranges from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

Preparation of the Vehicle Control
The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

Blank
On each plate three blank wells were tested (no cells and no treatment).

Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. 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.

Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Calculations were performed according to the OECD TG 442D.
Positive control results:
Experiment 1 : The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.52 and the EC1.5 125 µM.
Experiment 2 : The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.67 and the EC1.5 60 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Test substance - Experiment 1
Value:
1.04
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Imax
Run / experiment:
Test substance - Experiment 2
Value:
1.09
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Experiment 1
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• TMAO anhydrous showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with TMAO anhydrous. The Imax was 1.04 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.52 and the EC1.5 125 µM.

Experiment 2
• No precipitation was observed at the start and end of the incubation period in the 96-well plates.
• TMAO anhydrous showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
• No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with TMAO anhydrous. The Imax was 1.09 and therefore no EC1.5 could be calculated.
• The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.67 and the EC1.5 60 µM.

Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (125 µM and 60 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.52-fold and 2.67-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 12.8% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Table1          
Overview Luminescence Induction and Cell Viability of TMAO anhydrous in Experiment 1 and 2

Concentration(µM)

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

2000

Exp1 luminescence

1.01

1.00

0.96

0.98

0.98

0.96

0.96

0.95

0.97

1.00

1.03

1.04

Exp1 viability(%)

113.9

94.8

85.6

87.5

79.5

84.8

80.6

83.3

85.3

90.6

100.3

115.6

Exp2 luminescence

1.06

1.03

1.09

1.04

1.05

0.98

1.01

1.06

1.03

1.07

1.09

1.03

Exp2 viability(%)

100.9

109.3

110.4

103.3

112.4

110.9

113.1

110.7

103.1

106.5

85.4

104.3

 

Table2          
Overview Luminescence Induction and Cell Viability Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp1 luminescence

1.07

1.15

1.15

1.36

1.50***

2.52***

Exp1 viability (%)

99.6

101.2

106.6

108.7

115.8

86.8

Exp2luminescence

1.03

1.13

1.22

1.53***

1.92***

2.67***

Exp2viability(%)

108.9

117.0

118.9

124.7

124.2

129.2

***p<0.001 Student’s t test

 

Table3          
Overview EC1.5,Imax, IC30and IC50Values

 

EC1.5(µM)

Imax

IC30(µM)

IC50(µM)

Test item Experiment 1

NA

1.04

NA

NA

Test item Experiment 2

NA

1.09

NA

NA

PosControl Experiment 1

125

2.52

NA

NA

PosControl Experiment 2

60

2.67

NA

NA

NA = Not applicable


 

 

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, TMAO anhydrous is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions according to CLP criteria.
Executive summary:

The objective of this GLP compliant in vitro study was to evaluate the ability of TMAO anhydrous to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay according to OECD TG 442D method.

Batch 160405 of TMAO anhydrous was a white powder.  TMAO anhydrous was dissolved in dimethyl sulfoxide at 200 mM.  From this stock 11 spike solutions in DMSO were prepared.  The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series).  The highest test concentration was the highest dose required in the current guideline.  No precipitate was observed at any dose level tested.  Two independent experiments were performed.

Both experiments passed the acceptance criteria:

•       The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.  

•       The EC1.5 of the positive control was between 5 and 125 µM (125 µM and 60 µM in experiment 1 and 2, respectively).  A dose response was observed and the induction at 250 µM was higher than 2-fold (2.52-fold and 2.67-fold in experiment 1 and 2, respectively).

•       Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (6.5% and 12.8% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.  

TMAO anhydrous showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.  The maximum luciferase activity induction (Imax) was 1.04-fold and 1.09-fold in experiment 1 and 2 respectively.  TMAO anhydrous is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

In conclusion, TMAO anhydrous is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

DPRA (OECD TG 442C)

The objective of this in chemico GLP compliant study was to determine the reactivity of TMAO anhydrous towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) according to OECD TG 442C method.

After incubation of the test item with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm.  SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test item and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study.  

In the cysteine reactivity assay the test item showed 20.4% SPCC depletion while in the lysine reactivity assay the test item showed 0.2% SPCL depletion.  The mean of the SPCC and SPCL depletion was 10.3% and as a result the test item was considered to be positive in the DPRA and classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid.  TMAO anhydrous was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

KeratinoSens assay (OECD TG 442D)

The objective of this GLP compliant in vitro study was to evaluate the ability of TMAO anhydrous to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay according to OECD TG 442D method.

TMAO anhydrous was dissolved in dimethyl sulfoxide at 200 mM.  

TMAO anhydrous showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.  The maximum luciferase activity induction (Imax) was 1.04-fold and 1.09-fold in experiment 1 and 2 respectively.  TMAO anhydrous is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

In conclusion, TMAO anhydrous is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions.

U-Sens™ assay (OECD TG 442E)

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 dissolved in complete medium at 0.4 mg/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.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Three Klimisch-1-rated in-vitro studies on skin sensitisation are available with TMAO:

- OECD TG 442C: positive in the DPRA and classified in the “low reactivity class”

- OECD TG 442D: negative in the KeratinoSens assay

- OECD TG 442E: negative in the U-Sens™ assay

Based on a weight of evidence it is concluded that TMAO is not classified for skin sensitisation according to the CLP criteria.

No data is available by inhalation.