Trisodium bis[4-[4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzene-1-sulphonato(3-)]cobaltate(3-)

Endpoint summary

Description of key information

human Cell Line Activation Test (h-CLAT): negative

ARE-Nrf2 Luciferase Test: negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 30th to May 25th, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

FORMULATION
First, the test item was tried to be formulated in sodium chloride solution (saline). At the concentration of 100 mg/mL a total dissolution of the test item was observed with approximately 3 minutes of vortexing and 3 minutes of sonication and a rust color stable suspension was formed.
Then DMSO was tried at 500 mg/mL, but at this concentration only partial dissolution of the test item was observed after approximately 3 minutes of vortexing and sonication and undissolved particles were sunk to the bottom of the test tube. With additional amount of DMSO, the test item was tried to be dissolved at 250 mg/mL, however still no total dissolution could be observed. By going down another 2-fold, at 125 mg/mL the same result as before was obtained. No further dilution of the test item was attempted.
Since the formulation with saline was suitable for the study and the obtained concentration was higher than any concentration that could be gained with DMSO, moreover saline is the first preferred solvent for the test item, it was chosen as the appropriate solvent of the test item.

POSITIVE CONTROL
1-Chloro-2,4-dinitrobenzene

CELL / TEST SYSTEM
- Cells: THP-1 cell line, Human Acute Monocytic Leukemia
- Supplier: ATCC. The original cells (TIB-202) were subcultured into prepared cell lines (master cultures-MCs) in the testing laboratory.
- Maintance: RPMI-1640 modified medium (with 25 mM HEPES buffer) supplemented with 10 (v/v) % fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2.05 mM L-glutamine solution and 0.05 mM 2-mercaptoethanol.
- Preparation of the cells for the test: For testing, THP-1 cells were seeded at a density of either 0.1 × 106 cells/mL or 0.2 × 106 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintenance medium at 2 × 106 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 μL cell suspension / well (1 × 106 cells/well).

PRELIMINARY TESTS
Preparation master and working solutions
Preparation master and working solution were prepared with saline as follows: Eight master solutions (eight concentrations) were prepared of the test item stock solution, by two-fold serial dilutions using saline. In order to be able to determine CV75 value more precisely the concentration range was lowered in the second preliminary run. These master solutions were then further diluted 50-fold into culture medium to obtain the working solutions (WS).
The working solutions were finally used for exposure by adding an equal volume of working solution (500 μL) to the volume of THP-1 cell suspension (500 μL) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item.

Solvent/vehicle control
The solvent/vehicle control used for the test item and the positive control were culture medium and 0.2 % DMSO.

Test item exposure
The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated for 24 ± 0.5 hours at 37° C under 5 % CO2. The plates were sealed with microplate covers prior to the incubation to avoid evaporation of test item.

PI Staining
After 24 ± 0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample.

Cytotoxicity measurement by flow cytometry and estimation of CV75 value
The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Cell viability was analyzed by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.

MAIN TEST

Concentrations: 71.3, 59.4, 49.5, 41.3, 34.4, 28.7, 23.9 and 19.9 μg/ml

Test item dilutions
Saline was used to dissolve the test item for stock solution (SS) in the main tests, as well. The test item was first diluted to the concentration corresponding to the CV75 × 1.2 value (71.3 μg/mL) determined in dose finding assay. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using saline (eight concentrations). The master solutions were then further diluted 50-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate.

Solvent/vehicle controls
Culture medium was used as negative control for the test item and to assess the impact of DMSO. DMSO was tested as a solvent control for the positive control at a single final concentration in the plate of 0.2 %, so it underwent the same dilution as described for the working solutions.

Positive control
DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells.

Application of test item and control substances
Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 106 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours.

Fluorescein Isothiocyanate (FITC) staining
After 24 ± 0.5 hours of exposure, cells were transferred from the 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (250 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at approximately 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes.
After centrifugation (250 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at approximately 4° C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry.

Flow cytometry measurement and relative fluorescence intensity (RFI) determination
The expression of CD86 and CD54 is analysed with flow cytometry with the acquisition channel FL-1. Cytotoxicity was checked based on the PI uptake as in the preliminary tests and was analysed with the acquisition channel FL-3.
A total of 10,000 living cells (PI negative) are acquired (when the cell viability was low, up to 30,000 cells including dead cells could be acquired). Alternatively, data could be acquired for one minute after the initiation of the analysis. The cell viability was automatically calculated by the cytometer analysis program.
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 marked positive control (ctrl) and chemical-treated cells were calculated.

ACCEPTANCE CRITERIA
- The cell viabilities of medium and solvent/vehicle controls should be higher than 90 %.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and cell viability should be more than 50 %.
- In the solvent controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 > 150 % and CD54 > 200 %).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105 %.

Abnormal values
- RFI values cannot be less than zero. Regardless of the reason, such values should be omitted from the prediction.
- If an abnormal value is observed, check whether there are abnormal conditions in the run and record them in the reporting section.

Requirement for data acceptance
- For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
- For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90 %. When the test item is tested at 5000 μg/mL in saline (maintenance medium alternatively) or 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration, a negative result is acceptable even if the cell viability is above 90 %.

Run / experiment:

other: 2 out of 3 runs

Parameter:

other: % RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2 out of 3 runs

Parameter:

other: % RFI of CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

MFI, RFI AND VIABILITY VALUES OF CD86 AND CD54 MEASUREMENTS

	MFI (geom mean)			Corrected MFI			RFI (CD86)	RFI (CD54)			viability	MFI > 105 % to IgG
	CD 86	CD54	isotype	CD86	CD54					IgG		CD86	CD54
1st run
Control	5593	4344	3790	1803	554	100			100	94.8		148%	115%
DMSO 0.2 %	5084	4421	3497	1587	924	88			167	94.3		145%	126%
DNCB 4.0 µg/ml	13483	7522	4094	9389	3428	592			371	78.4
Test item 71.0μg/ml	3564	3974	3257	307	717	17			129	77.6
Test item 59.2 μg/ml	3987	4112	3331	656	781	36			141	78.9
Test item 49.3 μg/ml	3852	4273	3478	374	795	21			144	78.8
Test item 41.1 μg/ml	4124	4116	3057	1067	1059	59			191	77.7
Test item 34.2 μg/ml	4617	4329	3640	977	689	54			124	83.2
Test item 28.5 μg/ml	4365	4362	3535	830	827	46			149	86.8
Test item 23.8 μg/ml	4496	4222	3623	873	599	48			108	87.6
Test item 19.8 μg/ml	4656	4210	3795	861	415	48			75	87.9
2nd run
Control	4456	3854	3101	1355	753	100			100	93.7		144%	124%
DMSO 0.2 %	5143	3583	2852	2291	731	169			97	94.2		180%	126%
DNCB 4.0 µg/ml	11890	6956	3044	8846	3912	386			535	75.2
Test item 72.0 μg/ml	3760	3756	3096	664	660	49			88	76.8
Test item 60.0 μg/ml	3503	3699	3002	501	697	37			93	74.2
Test item 50.0 μg/ml	4124	3738	2968	1156	770	85			102	79.9
Test item 41.7 μg/ml	4038	3803	2861	1177	942	87			125	81.9
Test item 34.7 μg/ml	3805	3746	3023	782	723	58			96	82.6
Test item 28.9 μg/ml	3694	3752	2959	735	793	54			105	85.8
Test item 24.1 μg/ml	4062	3707	3041	1021	666	75			88	87.3
Test item 20.1 μg/ml	4308	3768	2951	1357	817	100			109	88.3
3rd run
Control	5705	4128	3385	2320	743	100			100	93.3		169%	122%
DMSO 0.2 %	5356	4117	3361	1995	756	86			102	92.3		159%	122%
DNCB 4.0 µg/ml	9058	5748	3305	5753	2443	288			323	80.6
Test item 71.0 μg/ml	4433	4237	3330	1103	907	48			122	80.5
Test item 59.2 μg/ml	4768	4191	3268	1500	923	65			124	81.3
Test item 49.3 μg/ml	4477	4302	3283	1194	1019	51			137	82.5
Test item 41.1 μg/ml	4736	4310	3294	1442	1016	62			137	79.5
Test item 34.2 μg/ml	4442	4296	3312	1130	984	49			132	85.0
Test item 28.5 μg/ml	4505	4043	3210	1295	833	56			112	87.3
Test item 23.8 μg/ml	4809	4107	3144	1665	963	72			130	87.0
Test item 19.8 μg/ml	5487	4110	3213	2274	897	98			121	88.4

DOSE FINDING TEST RESULTS

Test dose (µg/ml)	7.8	15.6	31.3	62.5	125.0	250.0	500.0	1000.0
Viability (%)	92.9	90.8	83.0	78.9	59.5	40.7	37.1	29.1
Test dose (µg/ml)	2.0	3.9	7.9	15.8	31.5	63.0	126.0	252.0
Viability (%)	96.2	95.6	94.3	90.6	85.8	67.1	54.2	41.4

Interpretation of results:

other: not sensitising

Conclusions:

The test item demonstrated an in vitro non-sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Executive summary:

The skin sensitization potential of the substance was studied in in vitro skin sensitisation human cell line activation test, according to OECD 442E.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. Based on their result, eight doses between 72.0 μg/mL – 19.8 μg/mL were used for the main test in three independent runs out of which two runs were concluded valid.

The increase in CD86 marker expression (RFI) was lower than 150 % at all tested doses (with > 50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the two valid negative results out of two valid runs, CD86 marker expression was concluded to be negative. Therefore, effective concentration for CD86 expression (EC150) was not determined.

The increase in CD54 marker expression (RFI) was lower than 200 % at all tested doses (with > 50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the two valid negative results out of two valid runs, CD54 marker expression was concluded to be negative. Therefore, effective concentration for CD54 expression (EC200) was not determined.

Since the CD86 and CD54 markers gave negative results in both valid runs, the overall h-CLAT prediction was concluded to be negative, as well.

Conclusion

Based on these results and the h-CLAT prediction model, the test item demonstrated an in vitro non-sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Cell or Test System (KeratinoSens™ cell line)
The cells used in this assay were the transgenic cell line KeratinoSens with a stable inserton of the luciferase construct supplied by Givaudan Schweiz AG . The cells were stored in vapor phase of liquid nitrogen. The original cells were propagated and subcultured into prepared cell line stocks (master cultures - MCs) in the 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.

KeratinoSens™ Master culture
Subcultured MC4 master culture was used for the study.

Formulation of the Test Item
Possible solvents for the test item were dimethyl sulfoxide (DMSO) and sterile ultrapure water or exposure medium.
The test item was formulated and examined as follows: first, solubility of the test item was evaluated in DMSO at the final concentration of 200 mM. The test item could be properly dissolved in DMSO after 5 minutes of vortexing and being in ultrasonic bath for another 5 minutes and an easy flowing, brownish and homogenous solution was gained. With ultrapure water the test item and solvent formulated a heavy flowing suspension.

Test item Master Solutions
Based on the test item stock solutions made of DMSO, 2-fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test item 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 240 µL exposure medium.

Positive controls
The positive control used was Trans-Cinnamaldehyde for which 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 controls
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 in 6.3.1, so that the final negative (solvent) control concentration was 1 % DMSO in exposure medium on the treated plates.This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO used in the tested chemical and in the positive control.

Preparation of cells
Cells were subcultured upon reaching 80 - 90 % confluence 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 (10 000 cells/well) homogenously. 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 item 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 item.

Luciferase activity measurements
After the 48-hour exposure time with the test item and control substances, cells were washed with DPBS two times (2 × 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.

Cytotoxicity
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 solubilised 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 item 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 should be performed. Each independent test was to be performed on a different day with fresh stock solution of test items and independently harvested cells. Cells may however have come from the same passage. 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 substance (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 fold. 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.

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.

Furthermore, the cellular viability is measured at the lowest concentration leading to ≥ 1.5-fold luciferase induction. This concentration should induce no significant cytotoxic effects and be below the IC30 value. In addition, viability should be > 70 % in at least two consecutive concentrations, otherwise the concentration range needs adjustment.

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at several concentrations in both tests. The EC1.5 values of the positive control fell between 7 µM and 30 µM (9 µM in both valid tests). The average inductions in the parallel plates for Trans Cinnamaldehyde at 64 μM were 83.16 fold and 38.56 fold in the first and second tests, respectively.

Run / experiment:

other: Test 1

Parameter:

other: Imax

Remarks:

(fold)

Value:

6.91

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Test 1

Parameter:

other: IC50

Remarks:

µM

Value:

417

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Test 1

Parameter:

other: IC30

Remarks:

µM

Value:

182

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Test 1

Parameter:

other: viability

Remarks:

%

Value:

70

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Test 1

Parameter:

other: EC1.5

Remarks:

(fold)

Remarks on result:

other: Since the values over 1.5 fold were not significant, EC1.5 value was not determined

Run / experiment:

other: Test 2

Parameter:

other: Imax

Remarks:

µM

Value:

4.05

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Test 2

Parameter:

other: IC30

Remarks:

µM

Value:

145

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Test 2

Parameter:

other: IC50

Remarks:

µM

Value:

245

Negative controls validity:

valid

Positive controls validity:

not valid

Run / experiment:

other: Test 2

Parameter:

other: EC 1.5

Remarks:

µM

Value:

513

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Test 2

Parameter:

other: viability

Remarks:

%

Value:

70

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

RESULTS
First test:
The test item induced cytotoxicity (or viability below 70 %) compared to the solvent/vehicle control over the concentration of 125 µM in KeratinoSens™ cells. Thus, IC30 and IC50 values were calculated as 182 µM and 417 µM.
The luciferase activity induction did not statistically significantly exceed the threshold of 1.5 fold compared to the respective negative control. The maximal fold induction (Imax) was 6.91 fold, though. Since the values over 1.5 fold were not significant, EC1.5 value was not determined. However, overall dose response could be observed.
No precipitation was observed at any point during the first test.
Based on the prediction model and the above described results, the test item was concluded negative in the first test, since the conditions for a positive result were not met.

Second (repeated) test:
The second test when conducted for the first time, did not meet all acceptance criteria concerning the positive control due to the EC1.5 value being lower than 7 µM. Therefore the test results were not included in the Report and the test was repeated with the same conditions.
The test item induced cytotoxicity (or viability below 70 %) compared to the solvent/vehicle control over the concentration of 125 µM in KeratinoSens™ cells. Thus, IC30 and IC50 values were calculated as 145 µM and 245 µM.
Although the luciferase activity induction exceeded the threshold of 1.5 fold compared to the respective negative control at the concentration of 1000 µM, the cell viability did not reach 70% at this concentration. The maximal fold induction (Imax) was 4.05 fold. EC1.5 value could was determined as 513 µM. An overall dose response could be observed.
No precipitation was observed at any point during the second test.
Based on the prediction model and the above described results, the test item was concluded negative in the second valid test, since no all conditions for a positive result were met.

Interpretation of results:

other: non sensitising

Conclusions:

The test item is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

The skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method), according to OECD guideline 442D.

In order to derive a prediction for the test item the results of two independent tests were used. Since the results of the two tests were concordant, a third was not needed in order to derive a conclusion. However, the second test was needed to be repeated due to not fulfilling all validity criteria for the positive control.

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

For the test item, twelve doses ranging from 2000.00 µM to 0.98 µM were used in both tests.

The test item induced cytotoxicity (a substance is considered cytotoxic if the viability is below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle at several concentrations in both tests. Thus, IC30 or IC50 values were determined and stated as 182 µM and 417 µM for the first test while 145 µM and 245 µM in the second valid test respectively.  

Although there were induction values exceeding the 1.5-fold threshold, they were either not statistically significant or not paired with viability values over 70 %. Therefore both valid tests were concluded negative since no all criteria were met for the positive respsonse.

Based on these results and the KeratinoSens™ prediction model, the test item is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Acid Brown 303 was tested for the assessment of skin sensitisation potential with ARE-Nrf2 Luciferase Test (KeratinoSens) according to OECD guideline 442D and in vitro Human Cell Line Activation Test (h-CLAT) according to the OECD guideline 442E.

ARE-Nrf2 Luciferase Test (KeratinoSens)

The skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method), according to OECD guideline 442D.

Two independent tests were used. Since the results of the two tests were concordant, a third was not needed in order to derive a conclusion. The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde, was statistically significant above the threshold of 1.5-fold in all tests.

The test item induced cytotoxicity (viability is below 70 %) in KeratinoSens™ cells compared to the solvent/vehicle at several concentrations in both tests. Thus, IC30 or IC50 values were determined and stated as 182 µM and 417 µM for the first test while 145 µM and 245 µM in the second valid test respectively.  

Although there were induction values exceeding the 1.5-fold threshold, they were either not statistically significant or not paired with viability values over 70 %. Therefore both valid tests were concluded negative since no all criteria were met for the positive respsonse.

Based on these results and the KeratinoSens™ prediction model, the test item is concluded negative for skin sensitization potential.

in vitro Human Cell Line Activation Test (h-CLAT)

The skin sensitization potential of the substance was studied in in vitro skin sensitisation human cell line activation test, according to the OECD guideline 442E.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests.

The increase in CD86 marker expression (RFI) was lower than 150 % at all tested doses (with > 50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the two valid negative results out of two valid runs, CD86 marker expression was concluded to be negative. Therefore, effective concentration for CD86 expression (EC150) was not determined.

The increase in CD54 marker expression (RFI) was lower than 200 % at all tested doses (with > 50 % of cell viability) compared to the respective negative controls in all valid runs. Based on the two valid negative results out of two valid runs, CD54 marker expression was concluded to be negative. Therefore, effective concentration for CD54 expression (EC200) was not determined.

Since the CD86 and CD54 markers gave negative results in both valid runs, the overall h-CLAT prediction was concluded to be negative, as well.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), skin sensitiser means a substance that will lead to an allergic response following skin contact.

The skin sensitising potential of Acid Brown 303 was evaluated taking into account experimental findings related to different aspects of skin sensitisation.

Two different key events of skin sensitisation were assessed by 2 specific in vitro test methods:

- key event 1 of skin sensitisation - KeratinoSens test (OECD 442D): negative

- key event 2 of skin sensitisation - hCLAT (OECD 442E): negative

Based on existing studies the substance is not classified as skin sensitizer according to CLP criteria set out in Regulation EC 1272/2008 (CLP).