p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 August - 02 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 18 June 2019 (Corrected 26 June 2020)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: SANCO Guideline 3030/99

Version / remarks:

rev.4; July 11, 2000. Technical Material and Preparations: Guidance for generating and reporting methods of analysis in support of pre- and post-registration data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive 91/414

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX) Protocol 154: Direct Peptide Reactivity assay (DPRA) for skin sensitisation testing

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

impurity 1

Reference substance name:

Methanol

EC Number:

200-659-6

EC Name:

Methanol

Cas Number:

67-56-1

Molecular formula:

CH4O

IUPAC Name:

methanol

impurity 2

Reference substance name:

Acetone

EC Number:

200-662-2

EC Name:

Acetone

Cas Number:

67-64-1

Molecular formula:

C3H6O

IUPAC Name:

acetone

impurity 3

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Constituent 1

Reference substance name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

EC Number:

269-198-6

EC Name:

p-nitrophenyl dihydrogen phosphate, compound with 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Cas Number:

68189-42-4

Molecular formula:

C6H6NO6P.2C4H11NO3

IUPAC Name:

4-nitrophenyl dihydrogen phosphate - 2-amino-2-(hydroxymethyl)propane-1,3-diol (1:2)

Test material form:

solid

Details on test material:

Batch / Lot No. 46587100

Specific details on test material used for the study:

Batch.No.: 46587100
Expiry date: 31 March 2022
Storage: refrigerator (2-8°C)

In chemico test system

Details on the study design:

Principle of the DPRA Method
The reactivity of a test chemical and synthetic Cysteine or Lysine containing peptides is evaluated by combining the test chemical with a solution of the peptide and monitoring the remaining concentration of the peptide following 24 hours of interaction time at room temperature. The peptide is a custom material containing phenylalanine to aid the detection and either Cysteine (“C”) or Lysine (“K”) as the reactive center.
Relative concentrations of the peptides following the 24 hour incubation are determined by high performance liquid chromatography with gradient elution and UV detection at 220 nm. Reaction samples, reference controls A, B and C, co-elution controls and positive controls are prepared and analysed in triplicates in batches of up to 26 chemicals (including controls) or a total of maximal 21 hours run.

Steps of the DPRA Method in chronological order
- Solubility assessment of the test chemical – ultrapure water was used as a solvent
- Preparation of buffer solutions
- Pre-weighting of test chemicals and positive control
- Pre-weighting of cysteine or lysine peptide for the stock solution
- Test chemical and positive control solution preparation
- Peptide stock solution preparation
- Serial dilution of standards
- Assembling of standards, reaction samples, positive controls, reference controls (A, B and C) and co-elution controls. For each set of control/sample replicates, the triplicate vials are prepared individually but from the same solutions.
- Preparation of HPLC system (column equilibration)
- HPLC analysis
- Data evaluation

Assembly of reaction samples and controls
• 1:10 ratio cysteine peptide (0.5 mM peptide, 5 mM test item)
- 750 µL cysteine peptide stock solution (or phosphate buffer for the co-elution control)
- 200 µL acetonitrile
- 50 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100mM positive control solution for the positive control)
• 1:50 ratio lysine peptide (0.5 mM peptide, 25 mM test item)
- 750 µL lysine peptide stock solution (or ammonium acetate buffer for the co-elution control)
- 250 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100 mM positive control solution for the positive control)

The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 22.5°C - 30°C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution. The batches were consisted of 2 parts: one part with the A reference controls, the calibration standards and the co-elution controls. These samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part. The other part contained the B and C reference controls, the positive control s and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended. The total HPLC analysis time should be less than 21 hours.

Formulation of the Test Item
The solubility of the test item was tested in a non-GLP preliminary solubility test as follows: the solubility of the test item was evaluated at the concentration of 100 mM. Acetonitrile was used first as a possible solvent but it did not dissolve the test item at all. The formulation separated into two phases after vortexing. However with ultrapure water, a homogenous and light yellow, clear solution was formed immediately at 100 mM.
The behavior of the formulation of the test item with ultrapure water was determined in the buffers of the test system (phosphate and acetate buffer) in a ratio corresponding to the reaction sample assembly. The compatibility of the formulation with the buffers of the test system was proven, no precipitate was observed in any cases and homogenous, clear solutions were obtained. Since this solvent is the first preferred vehicle after acetonitrile in the order of solvents listed in the guideline OECD TG 442C and the formulation complied with all obligations of the test guideline, no more formulations were checked but ultrapure water was chosen as the solvent.
The pre-experiments on solubility of the test item were not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of present study.

Synthetic Peptides
• Cysteine peptides
Name: Cysteine peptide
Batch no: 110116HS-MHeW0520-2
Storage: at -20°C or below
Purity: 95.8 %
Molecular weight: 750.88 g/mol
Sequence: Ac-RFAACAA-OH
Expiry date: November 28, 2020
Supplier: JPT Peptide Technologies GmbH

• Lysine peptide
Name: Lysine peptide
Batch no: 020517HS-MHeW0520-2
Storage: at -20°C or below
Purity: 93.09 %
Molecular weight: 775.91 g/mol
Sequence: Ac-RFAAKAA-OH
Expiry date: November 28, 2020
Supplier: JPT Peptide Technologies GmbH

HPLC System Conditions
HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
Serial number: L21445402951AE
Detector: 220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
Serial number: USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7µL
System equilibration: 50% phase A and 50% phase B for 2 hours at 30°C and running the gradient
twice before injecting the first sample
Run time: 20 min
Flow conditions: gradient flow
Mobile phases for HPLC:
Mobile Phase A – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

Evaluation method of experimental data
Percent peptide depletion
The concentration of the peptide was determined in each sample and positive control, from absorbance at 220 nm measuring the peak area of the appropriate peaks and calculating the concentration of the peptide using the linear calibration curves derived from the calibration standards.
The percent peptide depletion is determined in each reaction sample and positive control measuring the quotient of the peak area and the mean respective reference control C peak area, according to the formula described below:
peptide percent depletion = [1- { (peak area of the reaction sample or pc) / (mean peak area of reference controls C) } ] × 100

Presence of precipitate
If precipitation occurs it is recorded and caution is used in interpreting data. Samples can be centrifuged to settle and remove the precipitate to avoid clogging the HPLC. Centrifugation at low speed (max. 400 x g) is recorded as well.

Co-elution
In cases where a test chemical co-elutes with the lysine peptide, the Cysteine 1:10-only prediction model can be used. In cases where the test chemical co-elutes with the cysteine peptide and the peptide peak cannot be integrated, a determination of reactivity cannot be made based on the Percent Depletion data from the lysine reaction alone, and the data is reported as “inconclusive”. If the peak for the cysteine peak can be integrated, the instructions below are followed to determine an estimated Percent Depletion.

Negative depletion values
If the Percent Peptide Depletion is < - 10.0%, it is considered that this may be a situation of co-elution, inaccurate peptide addition to the reaction mixture or just baseline “noise.” If this happens, the co-elution controls are carefully analyzed. If the peptide peak appears at the proper retention time and has the appropriate peak shape, the peak can be integrated. The calculated percent peptide depletion is reported as an “estimate“. If this was only an issue for lysine, use the “cysteine-only” prediction model.
If this is an issue with cysteine or both cysteine and lysine, prediction is made based on the process below. If the peak does not have the proper shape due to complete overlap in retention time of the test chemical and peptide and cannot be integrated, calculation of Percent Peptide Depletion is not possible. If this is an issue for lysine, use the “cysteine-only” model. If this is an issue for cysteine or both cysteine and lysine, the data is reported as “inconclusive”.
Selection of prediction models:
no co-elution: Cysteine 1:10 / Lysine 1:10 prediction model
co-elution with only cysteine or cysteine and lysine peptides: inconclusive
co-elution with only lysine: Cysteine 1:10 prediction model

Co-elution Controls
If a chemical (Co-elution Control) absorbs at 220 nm and has a similar retention time as a peptide (Reference Control) and the peaks are overlapping, then co-elution of the test chemical with the peptide is reported.
In order to assure that baseline noise is not being called interference it is checked, if the “interfering” chemical peak has a peak area that is >10% of the mean peptide peak area in the appropriate Reference Control. If co-elution occurs, proper integration and calculation of Percent Peptide Depletion is not possible. The data is recorded as “interference” for that peptide.

Co-elution with reactivity and estimated depletion values
In some instances, a test chemical may have an overlapping retention time with either of the peptides and still be reactive with that peptide. This can make the peak area of the peptide appear to be larger than it really is, therefore the calculated percent depletion may be underestimated. If this is the case and the overlap in retention time between the test chemical and peptide is incomplete, percent depletion can still be calculated with a notation of “co-elution – percent depletion estimated”.

Results and discussion

Positive control results:

The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 68.89 % ± 1.44 % and with lysine peptide depletion value of 54.56 % ± 0.81 %.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

cysteine depletion

Remarks:

3 samples

Value:

3.27 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Remarks:

3 samples

Value:

0.21 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

Rejected Runs and Failure to Meet Acceptance Criteria
No runs were rejected.

Co-elution
No co-elution was observed with either of the peptides.
The range of retention time for cysteine peptide was between 8.394 and 8.505 while the range of retention time for lysine peptide was between 5.944 and 6.144.

System suitability
Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of A reference control sample replicates was 0.49 mM for the cysteine and 0.50 mM for the lysine peptide. A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions from the peptide stock solutions. Calibration standard points were analyzed by linear regression.
Means of the peak areas versus the concentrations of both peptides showed good linearity with r2 = 0.9981 for cysteine peptide and r2 = 0.9991 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM. All validity criteria were within acceptable limits and therefore the study can be considered valid.

Analysis sequences
Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion.
Moreover the CV % for the nine reference control B and C replicates in acetonitrile (acn) were smaller than the acceptable 15 % for both peptides, since it was 2.1 % and 0.2 % for cysteine and lysine peptides respectively. All validity criteria were within acceptable limits and therefore the study can be considered valid.

Cysteine and lysine depletion and mean peptide depletion of the test item
The mean cysteine and lysine peptide concentrations of the reference control C (solvent –ultrapure water) were within the acceptable 0.50 ± 0.05 mM range with 0.49 mM and 0.48 mM respectively.
The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 68.89 % ± 1.44 % and with lysine peptide depletion value of 54.56 % ± 0.81 %.
The mean cysteine peptide depletion value was 3.27 % ± 4.68 % while the lysine peptide depletion value of the test item was 0.21 % ± 0.21 %. When calculating the mean, negative depletion values are substituted with zero.

Assigning the test chemical to a reactivity class and category
The average percent peptide depletion was calculated for the test item. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 1.74 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class. The test item is classified as negative.

Applicant's summary and conclusion

Interpretation of results:

other: no or minimal reactivity towards the synthetic peptides

Conclusions:

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “4-NPP, di-TRIS” (CAS 68189-42-4) was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Executive summary:

In the course of this study the skin sensitization potential of the test item “4-NPP, di-TRIS” (CAS 68189-42-4) was studied using the Direct Peptide Reactivity Assay (DPRA).

For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and one with lysine peptides.

Peptide depletion resulting from the positive control cinnamaldehyde was within the expected percentage range both with cysteine and lysine peptides. Peptide depletion resulted from the positive control cinnamaldehyde was 68.89 % ± 1.44 % with cysteine peptide and 54.56 % ± 0.81 % with the lysine peptide.

The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine run (0.49 – 0.50 mM) and lysine run (0.48 – 0.50 mM) and the CV % for the nine reference controls B and C in acetonitrile were 2.1 % and 0.2 % percentages for the cysteine and lysine runs respectively. For each peptide, all validity criteria were met, confirming the validity of the study.

The mean cysteine peptide depletion value was 3.27 % ± 4.68 % while the lysine peptide depletion value of the test item was 0.21 % ± 0.21 %).

The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 1.74 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “4-NPP, di-TRIS” (CAS 68189-42-4) was concluded to be negative and to show no or minimal reactivity towards the synthetic peptides thus is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.