Reaction mass of tetrasodium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)] and tetraammonium [mono(4,4'-(1Z,1'Z)-(4,4'-dioxidobiphenyl-3,3'-diyl)bis(diazene-2,1-diyl)bis(5-amino-3-oxidonaphthalene-2,7-disulfonate(3-)) cuprate (4-)]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

other: read across from analogue substance

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM (INVITTOX) Protocol n°155: KeratinoSens™

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Direct Blue 267

IUPAC Name:

Direct Blue 267

In vitro test system

Details on the study design:

FORMULATION OF TEST ITEM
Test item could not be dissolved in DMSO after approximately 5 minutes of vortexing, but formed heterogeneous heavy flowing suspension. In ultrapure water, a partial dissolution of test item could be observed with undissolved particles floating in the solution. Thus, a less concentrated 100 mM solution was tried, with additional volume of ultrapure water a free flowing dark solution was gained. Since the overall volume of the above mentioned formulation did not allow proper visual inspection of homogeneity and was not sufficient for sterile filtering, a 10× volume was prepared at the same 100 mM concentration. This way a homogenous, free flowing solution was gained after 5 minutes of vortexing and was suitable for sterile filtration (through 0.22 micron filter membrane).
Since the formula fulfilled all requirements ultrapure water was chosen as the appropriate solvent of the test item.

CELLS USED
- Cell line: KeratinoSens™ cell line
- Description: immortalized adherent cell line derived from human keratinocytes (HaCaT) transfected with selectable plasmids.
- Supplier: Givaudan Schweiz AG
- Storage: vapor phase of liquid nitrogen.
- Subculturing: the original cells were propagated and subcultured into prepared cell line stocks (master cultures - MCs) in testing laboratory. Cells from this original stock could be propagated up to maximum 25 passages and were employed for routine testing using the maintenance/growth medium.

TEST CHEMICAL MASTER SOLUTION
Based on the test chemical stock solution made with ultrapure water at the highest soluble concentration (100 mM), two fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test chemical creating a 100 × master plate. The 100 × master concentrations were further diluted 25 fold into exposure medium to obtain the 4 × master plate, by adding 10 µl of the 100 × master concentrations to 230 µl exposure medium and correcting DMSO concentration by adding 10 µl DMSO to all twelve 4 × master solutions.

POSITIVE CONTROL
Trans-cinnamaldehyde, dissolved in Dimethyl sulfoxide (DMSO). A series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 µM.

NEGATIVE CONTROL
The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates.

PREPARATION OF THE CELLS
For testing cells were 80 - 90 % confluent and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium, and distributed into 96-well plates (10000 cells/well) homogenously; no sedimentation of the cells occurred during seeding. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.

EXPOSURE
After the 24 hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test chemical and control substances were added to each well in a way that an additional 4 fold dilution was achieved on the plate for the final concentrations to be established (50 µl of 4× master solution to 150 µl of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test chemical.

LUCIFERASE ACTIVITY MEASUREMENTS
After the 48 hour exposure time with the test chemical and control substances, cells were washed with DPBS (270 µl), and 1× lysis buffer (20 µl) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 µl) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

CITOTOXICITY
For the cell viability assay, medium was replaced after the 48 hour exposure time with MTT working solution (200 µl) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilized by the addition of isopropanol (50 µl). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

ACCEPTANCE CRITERIA
For each test chemical and positive control substance, in order to derive a prediction, at least two independent tests, each containing three replicates for the luminescence measurements and one for viability measurement, were needed. In case of discordant results between the two independent tests, a third test containing four replicates should be performed. Each independent test was to be performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may have come from the same passage however. KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the limitations stated in the OECD test guideline.

The luciferase activity induction obtained with the positive control, trans-cinnamaldehyde should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility or between 7 μM and 30 μM (based on the validation dataset). In addition, the average induction in the parallel plates for trans-cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of trans-cinnamaldehyde 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.

Historical control mean for EC1.5 value of the positive control: 14.6 ± 9.1 μM
Acceptance range for EC1.5 value of the positive control: 5.6 μM – 23.7 μM

Finally, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO should be below 20 % in each test which consists of 6 wells tested in triplicate.

Results and discussion

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

There was no statistically significant induction above 1.5-fold observed at any of the concentrations in either of the tests; therefore, no EC1.5 values were determined for the test item. The maximal fold induction was 1.29 for the first test and 1.34 for the second test. Based on the prediction model and the above described results, both tests were concluded negative.

There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at several concentrations in both tests. Cells of the last three wells were stained by the higher concentrations of the dark purplish - bluish test item solutions thus the resulted dark color may have affected absorbance measurement, and might gave a false sense of high survival rates. In the first test, the IC30 and IC50 values were 26 µM and 50 µM while in the second test 29 µM and 58 µM, respectively.

NEGATIVE CONTROL
The coefficient of variation (CV %) of the luminescence reading for the negative control DMSO was below 20 % in either of the tests (18.17 % and 18.86 % respectively). However one well of negative control (DMSO) values was omitted from the calculation as an outlier, since it is 25 % greater than the average of the other 5 values on the same plate in the second test.

POSITIVE CONTROL
The luciferase activity induction obtained with the positive control, trans-cinnamaldehyde was statistically significant above the threshold of 1.5 at four concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 6 µM and 9 µM in the two individual tests which fell into the corresponding acceptance range established based on the historical control data: 5.6 μM – 23.7 μM.
In addition, the average induction in the parallel plates for trans cinnamaldehyde at 64 μM was 28.35 and 83.88 fold. Although these values are out of the 2 – 8 fold induction range, the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control, therefore the tests were considered valid.

There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations in the first test however, there was cytotoxicity (viability < 70 %) induced by the positive control at 32 and 64 µM in the second test.

Any other information on results incl. tables

Average fold induction, significance and viability (%) values for the test item in the individual tests

Experiment 1

Concentration (uM)	0.5	1	2	4	8	16	31	63	125	250	500	1000
Plate 1	0.93	1.38	1.19	1.12	0.83	0.79	0.79	1.51	0.00	-0.02	-0.02	-0.02
Plate 2	1.21	1.13	1.5	1.01	0.9	0.74	0.74	1.33	0.00	-0.02	-0.02	-0.02
Plate 3	0.81	1.12	1.19	0.9	0.72	0.61	0.56	0.93	0.04	0.00	-0.01	-0.01
average induction	0.98	1.21	1.29	1.01	0.82	0.71	0.7	1.26	0.01	-0.02	-0.02	-0.02
significance	0.917	0.034	0.136	0.987	0.102	0.013	0.015	0.141	0.000	0.000	0.000	0.000
viability*	95 %	85 %	91 %	80 %	79 %	81 %	65 %	40 %	6 %	48 %	84 %	85 %

*Cells of the last three wells were stained by the higher concentrations of the test item solutions thus the resulted purple color may have affected absorbance measurement

Experiment 2

Concentration (uM)	0.5	1	2	4	8	16	31	63	125	250	500	1000
Plate 1	1.36	1.51	1.35	1.35	1.65	0.89	1.03	1.79	0.00	-0.03	-0.03	-0.03
Plate 2	1.06	1.03	1.03	0.81	0.81	0.35	0.58	1.31	-0.02	-0.06	-0.06	-0.06
Plate 3	0.89	0.83	1.01	1.01	1.29	0.57	0.68	0.92	-0.03	-0.05	-0.05	-0.05
average induction	1.10	1.12	1.13	1.05	1.25	0.61	0.76	1.34	-0.02	-0.04	-0.05	-0.05
significance	0.514	0.548	0.372	0.829	0.383	0.056	0.118	0.129	0.001	0.001	0.001	0.001
viability*	130 %	141 %	163 %	126 %	112 %	102 %	66 %	47 %	13 %	179 %	248 %	190 %

*Cells of the last three wells were stained by the higher concentrations of the test item solutions thus the resulted purple color may have affected absorbance measurement

Applicant's summary and conclusion

Interpretation of results:

other: test result is evaluated as part of a weight of evidence

Conclusions:

Based on the KeratinoSens™ prediction model, the test item was concluded negative, therefore, having a non-sensitizing potential under the experimental conditions.

Executive summary:

In the course of the study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test chemical and positive control substance, two independent tests were sufficient to derive a prediction, as test results were concordant and both tests met the acceptance criteria.

The luciferase activity induction obtained with the positive control, trans-cinnamaldehyde was statistically significant above the threshold of 1.5 at four concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 6 µM and 9 µM in the two individual tests respectively. In addition, the average induction in the parallel plates for trans cinnamaldehyde at 64 μM was 28.35 and 83.88 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations in the first test; however, there was cytotoxicity (viability < 70 %) induced at 32 and 64 µM in the second test.

For the test item twelve doses were used in two independent tests between 1000 µM to 0.5 µM.  There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at concentrations starting from 31 µM in both tests. In the first test, IC30 and IC50 values were 26 µM and 50 µM, while in the second test 29 µM and 58 µM respectively. However, the fold induction did not exceed the threshold of 1.5-fold at any concentrations compared to the respective negative controls in any of the independent tests. Therefore, both tests were concluded negative for luciferase gene induction.

Conclusion

Based on the KeratinoSens™ prediction model, test item was concluded negative, therefore, having a non-sensitizing potential under the experimental conditions.