Dioctyl phosphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 15th to September 1st, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

February, 2015

Deviations:

yes

Remarks:

Deviations according to protocol "LuSens Assay" provided by BASF SE.

Principles of method if other than guideline:

Determination of the sensitizing potential of the test item was performed with the "LuSens Test" on the basis to the protocol "LuSens Assay" provided by BASF SE.
The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay). The used cell line as well as the negative and the positive control is different in comparison to the OECD 442D guideline. Also the test performance differs in details even if the general procedure and the endpoint is similar to the one described in the BASF SE protocol.
LuSens test developed by the BASF SE is different from OECD 442D guideline for the following points:
- LuSens cell line was used;
- Cyto- toxicity Range Finder Test (CRFT) was performed;
- Dilution factor in the experiment was 1.2;
- Controls were tested at only one concentration;
- Ethylene glycol dimethylacrylate was used as positive control;
- During the experimental performance the luciferase induction was measured at a second 96-well plate;
- Regarding the acceptance criteria, the positive control must induce a luciferase induction of a minimum of 2.5 fold in comparison to the solvent control. In addition the viability must be ≥ 70 %. The negative control must induce a luciferase induction of < 1.5 fold and a viability of ≥ 70 %. Regarding the test item, a minimum of 3 test item concentrations has to be analysable (viability: ≥ 70 %).
Considering the demonstration of proficiency at laboratory facility (96 % results correctly categorized, more than 80 % according to OECD 442D), all deviations of the LuSens test in comparison to the OECD 442D were declared as uncritical.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyl phosphonate

EC Number:

217-315-6

EC Name:

Dioctyl phosphonate

Cas Number:

1809-14-9

Molecular formula:

C16H35O3P

IUPAC Name:

dioctyl phosphonate

Test material form:

liquid

In vitro test system

Details on the study design:

GENERAL PRINCIPLE:
This in vitro study was performed to assess the potential of the test item to activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” . It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway . In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two, but a maximum of three independent and valid experiments are performed.

PRELIMINARY SOLUBILITY TEST:
This test was performed to verify whether the test item is sufficiently soluble in DMSO. The solubility test was performed under non-GLP conditions. The test item was soluble at the required concentration of 200 mM.

PREPARATION OF STOCK SOLUTION:
Preparation was the same for all experiments. On the 1st day of the experiment, a stock solution (Citotoxicity range finding test -CRFT: 200 mM, experiments: 15.625 mM) of the test item in DMSO was prepared and 1:2.5 diluted. This dilution was afterwards used to prepare the geometric series of dilutions with DMSO (CRFT: factor 2, experiments: factor 1.2 ) of the resulting test item concentrations.

CONTROLS:
In accordance with the protocol of the BASF SE Ethylene glycol dimethylacrylate (EGDMA) was used as positive control and lactic acid as negative control. The controls were tested at only one concentration. The solvent control was DMSO.

TEST SYSTEM:
The LuSens cell line was specially designed for this test system by the BASF. It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2,Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck). For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of the laboratory to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells. For the Cytotoxicity Range Finder Assay cells of passage 9 were used. For the main experiments cells of passage 11 and 13 respectively, were used. After thawing the cells were cultivated in DMEM (9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

MEDIUM and CHEMICALS USED IN THE TEST:
-Chemicals used during the test: Ca2+/Mg2+-Solution for PBS, EDTA Solution (250 g/l), FCS Superior, Lysepuffer Glo Lysis Buffer 1X, MTT-Lysis Buffer SDS, MTT Solution (5 mg/ml), MTT-Working Solution, PBS, PBS + EDTA solution, PBS + Ca2+ / Mg2+, Steady-Glo® Reagent, Trypsin/EDTA, Isoton II.
-Medium: medium n.1 (DMEM alone, used only for the cell culture before starting the experimental phase), medium n.2 (DMEM 500 ml + FCS 50 ml) and medium n.3 (DMEM 500 ml + FCS 5ml)

CYTOTXICITY RANGE FINDER TEST:
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the main experiments. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. This yellow tetrazole is reduced to purple formazan in viable cells and can therefore be used for assessing the cell metabolic activity. A reduction of the viability below 70 % is defined as a cytotoxic effect. In the CRFT the following 12 nominal concentrations of the test item were tested: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1000 µM, 2000 µM. The exposure time was 48 h.
At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium n. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium n. 2. After quantification the cell suspension was adjusted to 83 000 (± 10 %) cells per ml. 120 µl of the cell suspension (= 10 000 cells) were seeded in a clear flat bottom 96 well plate. The plate was incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 24 h and 15 min. After the incubation time the medium was removed from the cells and 150 µl medium n. 3 was added to each well. Afterwards, 50 µl of the single test item concentrations as well as controls were added to the cells in triplicates (only test item concentrations). Twelve wells were used as solvent control, 6 wells were used as growth control, 3 wells were used as negative control and 2 wells were used as positive control. The plate was sealed with breathable tapes to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards, the plate was incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2. For the viability assay the MTT working solution was prepared by mixing 9 parts of medium n. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µl MTT working solution was added to each well. The plate was incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards, the solution was removed and 100 µl MTT-lysis buffer was added to each well. The plate was agitated for 5 min before it was measured at a wavelength of 570 nm and of 690 nm at the photometer. For calculation of the relative viability a validated Microsoft Excel file was used.

MAIN EXPERIMENTS:
Based on the results of CRFT the doses, the following 12 nominal concentrations were chosen for the main experiments : 8.4 µM, 10.1 µM, 12.1 µM, 14.5 µM, 17.4 µM, 20.9 µM, 25.1 µM, 30.1 µM, 36.2 µM, 43.4 µM, 52.1 µM, 62.5 µM.
At the time of seeding the cells were 90 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium n. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium n. 2. After quantification, the cell suspension was adjusted to 83 000 (±10 %) cells per ml. 120 µl of the cell suspension (= 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 25 h in both valid experiments. After the incubation time the medium was removed from the cells and 150 µl medium n. 3 were added to each well. Afterwards 50 µl of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium n. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.

EVALUATION OF THE VIABILITY IN THE MAIN EXPERIMENTS:
For the evaluation of the viability, one of the plates was used.
The MTT working solution was prepared by mixing 9 parts of medium n. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µl MTT working solution were added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 100 µl of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble formazan (purple color) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability.

EVALUATION OF THE LUCIFERASI INDUCTION IN THE MAIN EXPERIMENTS:
For the evaluation of the Luciferase induction, the second plate was used.
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 µl PBS (with Ca2+/Mg2+). Afterwards 100 µl per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 µl Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 µl per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer. For calculation of the luciferase induction as well as the relative viability a validated Microsoft Excel file was used.

DATA ANALYSIS (CALCULATION):
For calculation of result a validated Microsoft Excel file was used. All measured wells were corrected = OD570 Value - OD690 value.
Relative Viability = (Value of well of interest - blank) / (mean solvent control - blank) * 100
Afterwards the mean value of the single replicates was calculated.
The CV75-value (relative survival rate) was calculated by linear interpolation. This value is the substance concentration at which cell viability is 75 % compared to the control. Two concentrations were selected, one above 75 % viability and one below 75% viability as reference points. These data were used to calculate the slope as follows:
Slope = ( value rel. viability < 75 % - value rel. viability > 75 %) / (value μM of the rel. viability < 75 % - value μM of the rel. viability > 75 %).
Thereafter, the value of the Y intercept was calculated as follows:
Y intercept = value rel. viability < 75 % - (Slope* value μM of rel. viability < 75 % )
CV75 = ( 75- Y intercept) / Slope
Luciferase fold induction:
Fold induction = [ RLU value from measurement - RLU value from blank (only medium without cells)] /[Mean RLU value solvent - RLU value of blank (only medium without cells)]
Where RLU = Relative light unit
Mean fold induction (I max)= ( Fold induction of replicate 1+ Fold induction of replicate 2+ Fold induction of replicate 3) / 3

EVALUATION CRITERIA:
Each valid experiment (i.e. meeting all acceptance criteria, according to the procedure described above) is interpreted as follows:
- A test compound is considered to have sensitizing potential if the luciferase induction is above or equal to 1.5 fold compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic tested concentrations whereby at least three tested concentrations must be non-cytotoxic.
- A test compound is considered not to have sensitizing potential if the effects mentioned above are not observed.

ACCEPTABILITY:
- The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.
- The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70 %.
- The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.
- At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.

Results and discussion

In vitro / in chemico

Results

Key result

Run / experiment:

other: for all test item concentrations in the experiment II and in experiment III

Parameter:

other: luciferase induction fold compared to vehicle control

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:

MAIN EXPERIMENTS:
In total three independent experiments (experiment I, II and III) were performed. However, since no cytotoxicity was observed in experiment I (viability ≥ 97 %) an experimental error could not be excluded and the experiment was declared as invalid and was repeated. Experiment III was performed only to confirm the results of experiment II. 12 concentrations of the test item were tested. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment II and III: 8.4 µM, 10.1 µM, 12.1 µM, 14.5 µM, 17.4 µM, 20.9 µM, 25.1 µM, 30.1 µM, 36.2 µM, 43.4 µM, 52.1 µM, 62.5 µM. None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.
-Main experiment II: all control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 87 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 0.6 fold, negative control: 0.9 fold). However, the positive control induced a clear effect with an induction value of 2.8 fold in comparison to the sol-vent control. Cytotoxic effects were observed at the test item concentrations 62.5 µM to 36.2 µM. Therefore, those concentrations will not be included in the final evaluation. In all lower test item concentrations (30.1 µM to 8.4 µM) the viability was ≥ 86 %. In the Luciferase assay, none of the tested non-cytotoxic concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control. All values were ≤ 1.0 fold.
-Main experiment III: all control substances indicated the expected effect. No considerable reduction of the viability was detected (all values > 97 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 0.9 fold, negative control: 1.0 fold). However, the positive control induced a clear effect with an induction value of 3.7 fold in comparison to the solvent control. Cytotoxic effects were observed at the test item concentration 62.5 µM. Therefore, this concentration was not included in the final evaluation. In all lower test item concentrations (52.1 µM to 8.4 µM) the viability was ≥ 89 %. In the Luciferase assay, none of the tested non-cytotoxic concentrations induced a luciferase induction above the threshold of 1.5 fold in comparison to the solvent control. All values were ≤ 1.1 fold.
-Evaluation for both experiments: EGDMA (120 µM) was used as positive control and its luciferase induction is well within the historical data range of the positive control or within the laboratory historical 95% control limit in both experiments (values for positive control: 2.8 for exp. II and 3.7 for exp. III; historical data range : 3.8 - 14.7; 95% control limit : 2.53 - 9. 55). DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control in both experiments (values for negative control: 0.9 for exp. II and 1.0 for exp. III; historical data range : 0.8 - 1.3; 95% control limit : 0.86 - 1.14).

PRELIMINARY SOLUBILITY TEST
The test item was soluble at the required concentration of 200 mM in DMSO.

CYTOTOXICITY RANGE FINDER TEST (CRFT)
In the CRFT test no cytotoxic effect was observed at the controls as well as the test item concentrations 0.98 µM to 31.25 µM. The viability values were all ≥ 78 %. In all higher test item concentrations strong cytotoxic effects were detected (viability values ≤ 25 %). The CV 75 was calculated and was 33.2 µM.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The validity of the LuSens test at laboratory facility was demonstrated in a proficiency study. In this study, 22 proficiency chemicals (indicated by the OECD 442D guideline as well as the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS) were tested. As prescribed by the guidelines, more than 80 % (96 %) of the results were correctly categorized. Therefore, the proficiency of the LuSens test was demonstrated. For this reason, all deviations of the LuSens test in comparison to the OECD 442D are declared as uncritical. The end result is not affected by those changes.

ACCEPTANCE CRITERIA
All validity criteria were fulfilled:
- The average induction for the positive control was ≥ 2.5 fold and it had a relative viability of at least 70 %: Experiment II: Positive control : fold induction: 2.8; Relative viability: 87.1 %
Experiment III: Positive control : fold induction: 3.7; Relative viability: 97.9 %
- The induction triggered by the negative control and growth control were < 1.5 fold as compared to the induction of the solvent control and the viability was above 70 %:
Experiment II: Negative control : fold induction: 0.9; Relative viability: 105.9 %
Experiment II: Growth control : fold induction: 0.6; Relative viability: 125.3 %
Experiment III: Negative control : fold induction: 1.0; Relative viability: 110.7 %
Experiment III: Growth control : fold induction: 0.9; Relative viability: 139.8 %
- The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells was below 20 %:
Experiment II: 5.69 %
Experiment III: 10.32 %
- At least 3 test concentrations were within viability limits,i.e. have relative viability of at least 70 %:
Experiment II: 8 concentrations
Experiment III: 11 concentrations

EVALUATION OF THE RESULTS
In all tested concentrations of the test item for both experiments (II and III) no substantial and reproducible dose dependent increase of luciferase induction was measured . Dioctyl phosphonate does not have the potential to activate the Nrf2 transcription factor (no sensitizing potential) and it is not considered as a potential sensitiser.

Any other information on results incl. tables

Experiment II

		Induction ofLuciferase			Viability of the Cells
Parameter	Concen‑ tration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.06	5.69	100.0	6.08	6.08
Growth Control	-	0.6	0.06	10.03	125.3	7.31	5.84
Negative Control	5000	0.9	0.07	7.51	105.9	3.09	2.91
Positive Control	120	2.8	0.07	2.55	87.1	2.25	2.58
Test item	8.4	1.0	0.03	2.93	94.2	2.91	3.09
Test item	10.1	1.0	0.05	5.36	96.3	4.75	4.93
Test item	12.1	1.0	0.03	3.23	93.9	1.92	2.04
Test item	14.5	1.0	0.04	3.80	86.3	4.14	4.80
Test item	17.4	1.0	0.05	5.44	92.1	1.71	1.85
Test item	20.9	0.9	0.08	8.51	96.4	6.89	7.15
Test item	25.1	0.9	0.02	2.82	96.5	2.53	2.62
Test item	30.1	0.9	0.01	1.16	94.4	2.97	3.15
Test item	36.2	0.7*	0.18^*	25.42^*	50.0	18.45	36.89
Test item	43.4	0.5*	0.14^*	27.17^*	41.2	17.02	41.27
Test item	52.1	0.4*	0.05*	13.71*	33.3	10.12	30.36
Test item	62.5	0.2*	0.05*	26.47*	5.2	3.07	59.04

*high citotoxicity: value not used for evaluation

Experiment III

		Induction ofLuciferase			Viability of the Cells
Parameter	Concen- tration	Induction	Standard	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
			Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.10	10.32	100.0	3.40	3.40
Growth Control	-	0.9	0.04	4.44	139.8	4.98	3.56
Negative Control	5000	1.0	0.05	4.95	110.7	2.91	2.63
Positive Control	120	3.7	0.17	4.55	97.9	2.13	2.18
Test item	8.4	1.1	0.06	5.16	107.4	5.45	5.07
Test item	10.1	1.0	0.02	2.33	103.1	5.01	4.86
Test item	12.1	1.0	0.10	10.03	100.2	5.04	5.03
Test item	14.5	1.0	0.10	9.30	98.1	2.43	2.48
Test item	17.4	1.0	0.02	1.63	95.4	1.98	2.08
Test item	20.9	1.1	0.02	2.09	98.0	2.21	2.26
Test item	25.1	1.1	0.07	6.74	95.0	5.17	5.44
Test item	30.1	1.1	0.03	2.54	93.6	2.36	2.52
Test item	36.2	1.1	0.07	6.91	92.8	0.87	0.94
Test item	43.4	1.1	0.05	4.38	91.0	1.39	1.52
Test item	52.1	1.1	0.11	10.15	89.2	3.80	4.27
Test item	62.5	0.9*	0.05	5.84	60.8	5.09	8.38

*high citotoxicity: value not used for evaluation

Citotoxicity Range Finder Test

Parameter	Concentra- tion	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	[%]		[%]
Solvent Control	-	100.0	5.10	5.10
Growth Control	-	156.0	7.24	4.63
Negative Control	5000	103.0	2.18	2.12
Positive Control	120	90.0	0.69	0.78
Test item	0.98	111.5	1.12	1.01
Test item	1.95	104.0	2.14	2.06
Test item	3.91	102.2	1.90	1.86
Test item	7.81	101.3	0.14	0.14
Test item	15.63	94.2	1.75	1.86
Test item	31.25	78.3	8.19	10.46
Test item	62.5	24.7	12.93	52.30
Test item	125	5.6	8.58	154.18
Test item	250	5.6	7.55	134.65
Test item	500	2.0	3.72	185.29
Test item	1000	0.0	0.00	86.60
Test item	2000	0.0	0.00	0.00

Applicant's summary and conclusion

Interpretation of results:

other: not classified as skin sensitizer according to the CLP Regulation (EC n.1272/2008).

Conclusions:

Dioctyl phosphonate resulted to be negative (non-sensitizer) in Lu-Sens test.

Executive summary:

Dioctyl phosphonate was tested to evaluate its potential to activate the Nrf2 transcription factor by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens As-say). The assay differs in some points from the OECD guideline. Considering the demonstration of proficiency at laboratory facility (96 % results correctly categorized, more than 80 % according to OECD 442D), all deviations of the LuSens test in comparison to the OECD 442D were declared as uncritical.

The assay included a cytotoxicity range finder test (CRFT) and three independent experiments (experiment I, II and III) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the experiments were determined. Since no cytotoxicity was observed in experiment I (viability ≥ 98 %) an experimental error could not be excluded and the experiment was declared as invalid and was repeated.

In the experiments, the highest nominal applied concentration (62.5 µM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMSO (final concentration: 1 %) was used as solvent control and medium n. 3 as growth control. Lactic acid (5000 µM) was used as negative control and EGDMA (120 µM) as positive control.

No substantial and reproducible dose dependent increase in luciferase induction above 1.5 fold was observed in both experiments up to the maximal concentration of the test item.

Under the experimental conditions of this study, the test item Dioctyl phophonate was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).