Myo-Inositol, hexakis(dihydrogen phosphate), dodecasodium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

18.09.2017-13.10.2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read across is given in section 13 of IUCLID.
This endpoint study record is part of a Weight of Evidence approach comprising a read-across to three seperate studies (OECD 442C, 442D, 406) and two different supporting substances. All three data sources agree in that the test substances don`t have the portenital of a skin sensitizer. The data sources are sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

LuSens Assay submitted by BASF SE

Deviations:

yes

Remarks:

LuSens line was used; study was perfomred according to BASF SE protocol

Principles of method if other than guideline:

Deviations from the Guideline
The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is only based on the OECD 442D Guideline.
The deviations from the OECD 442D are given below:
1.For the test the LuSens cell line was used. This cell line was developed by the BASF SE.
2.In order to determine the concentration range applicable for experiment I and II a Cy-totoxicity Range Finder Test (CRFT) was performed. This test was performed in ac-cordance to the protocol of the BASF SE.
3.The dilution factor in the experiments is 1.2fold.
4.The controls are tested at only one concentration.
5.As positive control p-Phenylenediamine was used.
6.During the experimental performance the luciferase induction is measured at a second 96-well plate in accordance to the protocol of the BASF SE.
7.Regarding the acceptance criteria, the positive control must induce a luciferase induc-tion of a minimum of 2.5 fold in comparison to the solvent control. In addition the via-bility 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 con-centrations has to be analysable (viability: ≥ 70 %).
Prior to routine use, the validity of the LuSens test at LAUS GmbH 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 LU-CIFERASE 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.

The deviations were assessed and signed by the study director on 25. Oct. 2017.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

According to Comission Regulation 2016/1688 amending Annex VII to regulation 1907/2006

Test material

Test material information

Constituent 1

Reference substance name:

Myo-Inositol, hexakis(dihydrogen phosphate), sodium salt

EC Number:

238-242-6

EC Name:

Myo-Inositol, hexakis(dihydrogen phosphate), sodium salt

Cas Number:

14306-25-3

Molecular formula:

C6H18O24P6.xNa

IUPAC Name:

Esters of sodium hydrogen phosphate with myo-Inositol, plant-derived (old name: myo-Inositol, hexakis(dihydrogen phosphate), sodium salt)

Test material form:

solid: bulk

Details on test material:

- Appearance: white solid powder
- Homogeneity: homogenous
- Density: 2.0401 ± 0.0009 g/cm3 at 20 ± 0.2 °C
- Moisture content: 0.1 - 10% (water is an inherent constituent of the UVCB substance)

In vitro test system

Details on the study design:

VEHICLE CONTROL: Medium No. 3 (Biochrom GmbH)
POSITIVE CONTROL: p - Phenylenediamine
NEGATIVE CONTROL: D,L- Lactic acid
TEST SYSTEM:
- Cells used: LuSens Cell Line
- Supplier: BASF SE;Ludwigshafen, Germany
Preparation of cells:
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 no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 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).

PRELIMINARY STUDY:
-Cytotoxicity Assay
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 of the cell metabolic activity. A reduction of the viability below 70 % is defined as a cytotoxic effect.
In the CRFT 12 nominal concentrations (0.2µg/ml - 400µg/ml) of the test item were tested.
The prepared cell 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. Afterwards, 200 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 no. 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 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 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. According to the results the dose levels for the
main study were selected
MAIN STUDY:
The chemicals must be tested in two independents experiments at least.
Experiment I and II were performed in the same way. Both prepared cell plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere
for 24 h and 30 min in Experiment I and 24 h and 45 min in Experiment II.
The treatment procedure was performed on both 96 well plates identically:
After the incubation time the medium was removed from the cells and 200 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 no. 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.

- VIABILITY
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 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-(4,5-
Dimethyl 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

- LUCIFERASE INDUCTION:
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.

Test positive Criteria:
A test compound is considered to have the potential to activate the Nrf2 transcription factor 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.
Test negative Criteria:
A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the above effects are not observed.

Results and discussion

Positive control results:

Luciferase induction: 6.4/ 7.0 fold to solvent control
Relative Viability: 84.7/ 80.7%

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: Luciferase induction

Value:

0.416

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: Luciferase induction

Value:

0.394

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptability:
1. The average induction of the positive control was >= 2.5 and had a relative viability of >=70%
2. The induction triggered by the negative control and growth control was < 1.5 fold compared to the induction of the solvent control
and the viability was >= 70%
3.The average percentage standard deviation (luciferase induction) of the variability in at least21 solvent control wells were below 20 %.
4. More than 3 concentrations have been within viability limits, i.e. have relative viability of at least 70 %.
Thereby the acceptability (validity) criteria have been fulfilled

Any other information on results incl. tables

RESULTS

- Results of Experiment I

All control substances indicated the expected effect.

No considerable reduction of the viability was detected (all values ≥ 84 %).

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: 1.0 fold, negative control: 1.2 fold). However, the positive control induced a clear effect with an induction value of 6.4 fold in comparison to the solvent control.

No cytotoxic effect was observed at the test item concentrations 54 μg/mL to 333 μg/mL.

The viability values were all ≥ 70 % and therefore analysable for luciferase induction.

A slight cytotoxic effect was only observed at the highest concentration 400 μg/mL. Therefore, the result of this concentration is not included for the final evaluation.

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.

Results of Experiment II

All control substances indicated the expected effect.

No considerable reduction of the viability was detected (all values ≥ 80 %).

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: 1.0 fold, negative control: 1.2 fold). However, the positive control induced a clear effect with an induction value of 7.0 fold in comparison to the solvent control.

No cytotoxic effect was observed at the test item concentrations 54 μg/mL to 278 μg/mL.

The viability values were all ≥ 76 % and therefore analysable for luciferase induction.

Slight cytotoxic effects were only observed at the two highest concentrations 400 μg/mL and 333 μg/mL. Therefore, the results of those concentrations are not included for the final evaluation.

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.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line.

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 assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) 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 two experiments were determined.

In the experiments, the highest nominal applied concentration (400 μg/mL) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.21) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

Vehicle control (Medium no. 3), negative control ( Lactic acid) and postive control (p - Phenylenediamine) run in parallel

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

item.

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