Methyl dihydrogen phosphate

Endpoint summary

Description of key information

In vitro studies are ongoing and the results of two concordant studies will determine the overall prediction. However, based on chemical type, the substance is considered unlikely to cause skin sensitisation. ***AWAITING THIRD IN-VITRO STUDY***

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:

2 October 2018 - 31 January 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

yes

Remarks:

See Deviations text below.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: DPRA

Specific details on test material used for the study:

Batch 34448
Purity 67.9 - 71.2% Methyl dihydrogen phosphate
Expiry 30 March 2022

Details on the study design:

Preparation of test item
The concentration of the test item to be used was determined based on the molecular weight (MW) 96 g/mol and the purity, the purity value 67.9-71.2 % was treated as 67.9 %. The target weight (± 10 %) of the test item was calculated. 100 mM test item solution was prepared by dissolving 42.3 mg and 42.5 mg test item in 3 mL of the solvent acetonitrile for the Cys-peptide and Lys-peptide, respectively.

HPLC was used for the measurement of peptide depletion of the Cys-peptide and the Lys-Peptide.

Positive control
Positive controls were treated identically as the test item. The following positive controls were used:
• Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %, batch no. MKBT8955V) was used as 100 mM (± 10 %) solution in acetonitrile for the Cys-peptide.
• 2,3-Butanedione (CAS 431-03-8, ≥99 %, batch no. BCBS3560V) was used as 100 mM (± 10 %) solution in acetonitrile for the Lys-peptide

As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione was used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate. Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion.


Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.

Test solutions
Dilution buffers
• 2 mL Acetonitrile were mixed with 8 mL phosphate buffer, pH 7.498 (Peptide dilution buffer C)
• 2 mL Acetonitrile were mixed with 8 mL ammonium acetate buffer, pH 10.204 (Peptide dilution buffer K)

Peptide stock solutions
The peptide stock solutions were freshly prepared for each assay.
• 0.667 mM Cys-Peptide solution was prepared by dissolving 20.0 mg of the peptide in 40 mL phosphate buffer, pH 7.498. (batch no. 20181024)
• 0.667 mM Lys-Peptide solution was prepared by dissolving 25.9 mg of the peptide in 50 mL ammonium acetate buffer, pH 10.204. (batch no.20181009)

Peptide calibration standards
From each peptide stock solution, the following calibration standards were prepared in the appropriate dilution buffer: 0.534 / 0.267 / 0.1335 / 0.0667 / 0.0334 / 0.0167 mM Peptide. Calibration samples were analysed before the samples containing the test item. Blank dilution buffer was also measured.

Test item samples
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions. A final volume of 1 mL per sample was prepared for each sample.


Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 °C for 22 h and 5 minutes for the Cys-peptide and 22 h for the Lys-peptide, respectively. All three replicates for the Cys-peptide were turbid after incubation. They were centrifuged (10 min, 400 g) and only the clear supernatant was used for the measurement. None of the replicates for the Lys-peptide were turbid after incubation.

According to the test guideline, the reactivity is classified as “high”, “moderate”, “low” or “minimal” using the Cysteine 1:10/Lysine 1:50 prediction model shown in the table below.

Positive control results:

The mean peptide-depletion of the positive control 2,3 butanedione for the Lys-peptide was marginal out of the range of historical data, but nevertheless the value 40.25 % was within the range given in the OECD442C.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

0 %

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Value:

0 %

Other effects / acceptance of results:

No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

Calculated peptide depletion values for the Cys-Peptide

Sample name	Depletion (%)
	Single	Mean	SD
Positive control Rep 1	75.44	76.14	0.94
Positive control Rep 2	75.77
Positive control Rep 3	77.21
Test item Rep 1	0.00 (-1.07)	0.00	0.00
Test item Rep 2	0.00 (-1.34)
Test item Rep 3	0.00 (-1.23)

Negative depletion values were considered as “zero” when calculating the mean.

Calculated peptide depletion values for the Lys-Peptide

Sample name	Depletion (%)
	Single	Mean	SD
Positive control Rep 1	38.14	40.25	3.99
Positive control Rep 2	37.76
Positive control Rep 3	48.46
Test item Rep 1	0.00 (-3.33)	0.00	0.00
Test item Rep 2	0.00 (-0.73)
Test item Rep 3	0.00 (-0.92)

Negative depletion values were considered as “zero” when calculating the mean.

Conclusions:

The mean peptide depletion in the Cys-peptide and Lys-peptide assay was 0.00 %, therefore the test item was classified with:

DPRA Prediction: Negative Reactivity class: Minimal

Executive summary:

DPRA Prediction: Negative Reactivity class: Minimal

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 2018 to 16 august 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

according to Draft Guideline 7 July 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Batch 34448
Purity 67.9-71.2% Methyl dihydrogen phosphate
Expiry 30 March 2022

Details on the study design:

Preparation
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and medium (DMEM). The test item is soluble in both solvents at the required concentration (200 mg/mL). Since DMSO is the preferred solvent in the LuSens test, DMSO was used as solvent. Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mg/mL test item in DMSO was prepared. Subsequent dilution to 1% finally yielded a maximum concentration of 2000 µg/mL in the pre-test and the experiments. For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiments: factor 1.2) on a master plate. Afterwards all concentrations were further diluted (1:25) in medium no. 3 on a dilution plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the dilution plate to the corresponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

Test System
Reasons for the Choice of the LuSens Cell Line
The LuSens cell line was specially designed for this test system by the BASF SE (Ludwigshafen, Germany). 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).

Cell Cultures
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the cell bank of LAUS GmbH 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 10 were used. For both main experiments cells of passage 12 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.

PERFORMANCE OF THE STUDY
Dose Selection for Experiment I and II
In accordance with the OECD guideline 442D (draft version 7. July 2017) and the protocol of the BASF SE, the maximum final test item concentration should be 2000 µM. For a test chemical which has no defined molecular weight, the final test item concentration 2000 µg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility). In the case of a cytotoxic result, the concentrations for experiment I and II should be determined so that at least one of them is in the cytotoxic range. Since no cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations were chosen for experiment I and II: 269 µg/mL, 323 µg/mL, 388 µg/mL, 465 µg/mL, 558 µg/mL, 670 µg/mL, 804 µg/mL, 965 µg/mL, 1157 µg/mL, 1389 µg/mL, 1667 µg/mL, 2000 µg/mL. In the main experiments, a reduction of the viability below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction.

Experimental Parameters of Experiment I and II
Experimental Performance Experiment I and II were performed in the same way. Experiment II serves only to confirm the results of experiment I. 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). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 23 h 45 min in experiment I and 23 h 30 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 150 µL medium no. 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 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. 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 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 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 colour) to its insoluble formazan (purple colour) 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. 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.

Positive control results:

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 96 %). 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: 1.1 fold). However, the positive control induced a clear effect with an induction value of 3.7 fold in comparison to the solvent control.

Key result

Run / experiment:

other: I & II

Parameter:

other: Luuciferase induction fold

Value:

1.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Results of Experiment I
No cytotoxic effect was observed at any of the test item concentrations. In the Luciferase assay, the luciferase induction was ≥ 1.5 fold and statistically significantly increased in comparison to the solvent control in the test item concentrations 2000 µg/mL, 1667 µg/mL, 1389 µg/mL, 1157 µg/mL, 670 µg/mL and 269 µg/mL.

Results of Experiment II
No cytotoxic effect was observed at any of the test item concentrations. In the Luciferase assay, the luciferase induction was ≥ 1.5 fold and statistically significantly increased in comparison to the solvent control in all test item concentrations.

Conclusions:

The luciferase induction was ≥ 1.5 fold and statistically significant compared to the vehicle control in more than 2 consecutive non-cytotoxic tested concentrations in experiment I and II.

Therefore, the test item Methyl dihydrogen phosphate is considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential) under the conditions of the LuSens test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on chemical type, the substance is unlikely to cause skin sensitisation and classification under the criteria given by Regulation (EC) No. 2072/2008 is not required.