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

Endpoint summary

Description of key information

- DPRA (OECD 442C): mean peptide depletion: 1.74%, no or minimal reactivity, Non sensitizer.

-  KeratinosensTM(OECD 442D): Negative, Non sensitizer
Cytotoxic: no cytotoxicity observed.
Imax: 1.06 and 1.02 fold induction

-QSAR DEREK NEXUS version 6.1 did not yield any alerts for skin sensitization for the test item. 4-NPP, di-TRIS is predicted to be not sensitizing to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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

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)

Specific details on test material used for the study:

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

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”.

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 %.

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

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

25 August - 03 September 2020

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:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

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

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

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 3 minutes of vortexing. Then the test item was dissolved in ultrapure water, a total dissolution of the test item was observed with vortexing and a clear, yellow and homogenous solution was formed.
Since the formulation with the preferred vehicle, DMSO fulfilled all requirements, DMSO was chosen as the appropriate solvent of the test item in this study. The DMSO solution can be considered self-sterilising, so that no sterile filtration is needed.
The pre-experiments on solubility of the test item was 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.

Cell or Test System (KeratinoSens™ cell line)
The KeratinoSens™ cell line is a transgenic cell line with a stable Luciferase construct insertion.
Name: KeratinoSens™ cell line
Description: immortalised adherent cell line derived from human keratinocytes (HaCaT) transfected with selectable plasmids
Supplier: Givaudan Schweiz AG
Lot Number: 20160415
Date of production: April 15, 2016
Storage: Vapor phase of liquid nitrogen

KeratinoSens™ Master culture
Subcultured MC2 master culture was used for the study.
ID of the cell line: KeratinoSens™ MC4 p5 20180308-20200817
Date of preparation: March 08, 2018
Date of thawing: August 17, 2020
Passage number at start: p7
Passage number at the end: p9

Procedure of the KeratinoSens™ method
0. Preincubation of cells
1. Seeding of cells for testing - 24 h incubation
2. Preparation of the stock solution
3. Preparation of master plates
4. Exposure – 48 h incubation
5. Luciferase activation measurement
6. Cytotoxicity assessment

Principle of the KeratinoSens™ method
The KeratinoSens™ method is an in vitro assay that quantifies the extent of luciferase gene induction following 48 hours incubation time of the KeratinoSens™ reporter cells with the test items. Luciferase gene induction is measured in the cell lysates by luminescence detection using a light producing luciferase substrate (Luciferase Reagent). Cytotoxicity and the relative luminescence intensity of luciferase substrate in the lysates are measured and luciferase induction compared to solvent/vehicle control is calculated.
KeratinoSens™ cells were derived from HaCaT human keratinocytes and transfected with selectable plasmids containing luciferase gene under the transcriptional control of the AKR1C2 ARE gene sequence, upstream of the SV40 promoter. AKR1C2 is known to be one of the genes up-regulated upon contacting skin sensitisers in dendritic cells. Therefore, this method is able to mimic the activation of the Keap1-Nrf2-ARE regulatory pathway and is relevant for the assessment of the skin sensitisation potential of test items. A prediction model is used, to support the discrimination between sensitisers and non-sensitisers.

Preparation of the master plate
- 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 control
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 control
The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the test item master and working solution concentrations, 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 (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.

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 (14 µM and 8 µM in the first and second tests respectively).
The average inductions in the parallel plates for Trans-Cinnamaldehyde at 64 µM were 3.81 fold and 137.41 fold in the first and second tests, respectively.
Although the luciferase activity induction in the second test was outside of the 2 – 8-fold induction range, there was a clear dose response relationship in the luciferase activity induction with increasing concentrations and corresponding fold induction rise, therefore this result was accepted as valid.
In the first test, there was no cytotoxicity (or cell viability lower than 70 %) induced by the positive control at any of the tested concentrations. However, in the second test, the positive control induced cytotoxicity (or viability below 70%) compared to the solvent/vehicle control at the highest tested concentration (at 64 µM) in KeratinoSens™ cells.

Key result

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.06

Cell viability:

The test item induced no cytotoxicity (or viability below 70 %) compared to the solvent/vehicle control at any concentration in KeratinoSens™ cells.

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Coefficient of Variation (CV %): 7.65 %

Positive controls validity:

valid

Remarks:

Imax: 3.81

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.02

Cell viability:

The test item induced no cytotoxicity (or viability below 70 %) compared to the solvent/vehicle control at any concentration in KeratinoSens™ cells.

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

Coefficient of Variation (CV %): 10.75 %

Positive controls validity:

valid

Remarks:

Imax: 137.51

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

For the test item, twelve doses ranging from 2000.00 µM to 0.98 µM were used in both tests.
The test item induced no cytotoxicity (or viability below 70 %) compared to the solvent/vehicle control at any concentration in KeratinoSens™ cells. Thus, IC30 and IC50 values were not calculated.

The luciferase activity induction did not exceed the threshold of 1.5 fold compared to the respective negative control in any of the experiments. The maximal fold induction (Imax) was 1.06 fold and 1.02 fold in experiments 1 and 2, respectively. Therefore, EC1.5 value could not be determined. Moreover, no dose response could be observed).
No precipitation was observed at any point during the tests.

Each individual test met the acceptance criteria for the negative and positive controls and were
therefore considered valid under the conditions described.

Interpretation of results:

other: no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes

Conclusions:

Based on these results and the KeratinoSens™ prediction model, “4-NPP, di-TRIS” (CAS number 68189-42-4) is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

In the course of this study the skin sensitization potential of the test item “4-NPP, di-TRIS” (CAS number 68189-42-4) was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

For the test item and positive control substance, in order to derive a prediction two valid independent tests were conducted, in which the concluded results were concordant.

The luciferase activity induction obtained with the test item was not statistically significant above the threshold of 1.5 at any concentration in either test, meeting all acceptance criteria and the criteria for a negative response.

Since the results of the two valid tests were concordant, no more tests were needed. The overall result was concluded negative for luciferase gene induction.

Based on these results and the KeratinoSens™ prediction model, “4-NPP, di-TRIS” (CAS number 68189-42-4) is concluded negative for skin sensitization potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

22 June 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE: DEREK NEXUS
2. MODEL (incl. Version number): DEREK NEXUS 6.1.0.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: C1(=CC=C(C=C1)[N+]([O-])=O)OP(=O)(O)O.NC(CO)(CO)CO.NC(CO)(CO)CO
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL: See the QMRF in the study document attached
5. APPLICABILITY DOMAIN (OECD principle 3)
- Descriptor domain: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert or reasoning rule then Derek makes a negative prediction. The applicability of the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
- Structural fragment domain: For skin sensitization, which features multiple alerts believed to cover most of the mechanisms and chemical classes responsible for activity, “no alerts fired” may be extrapolated to a negative prediction. All structure fragments were found in the DEREK database and consequently the structure falls within the applicability domain of DEREK’s skin sensitization end point.
- Mechanism domain: As the prediction is “no alerts fired” none of the mechanisms for skin sensitization is predicted to be applicable to this structure.
- Metabolic domain: Not relevant.

6. ADEQUACY OF THE RESULT
- Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitization.
- Outcome: Substance should not be classified according to DEREK NEXUS; however,
this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for
covering the endpoint skin sensitization for registration under REACH.
- Conclusion: The result is adequate to be used in a weight-of-evidence approach
together with in chemico/in vitro studies to complete the endpoint skin sensitization.

Qualifier:

according to guideline

Guideline:

other: REACH guidance on QSARs R.6

Version / remarks:

Prediction on the potential for skin sensitization with the in silico model DEREK NEXUS, version 6.1

Deviations:

no

Principles of method if other than guideline:

Principles of method if other than guideline
- Software tool(s) used including version: in silico model DEREK NEXUS version 6.1.0
- Knowledge Base: Derek KB 2020 1.0
- Model description: see field 'Justification for non-standard information', 'Attached justification'
- Justification of QSAR prediction: see field 'Justification for type of information', 'Attached justification'

GLP compliance:

no

Specific details on test material used for the study:

SMILES: C1(=CC=C(C=C1)[N+]([O-])=O)OP(=O)(O)O.NC(CO)(CO)CO.NC(CO)(CO)CO

Key result

Parameter:

other: alerts for skin sensitation

Remarks on result:

other: No indication of skin sensitisation. DEREK NEXUS version 6.1 did not yield any alerts for skin sensitization of 4-NPP, di-TRIS (CAS 8189-42-4). 4-NPP, di-TRIS (CAS 8189-42-4) is p redicted to be not sensitizing to the skin.

Outcome of the prediction model:

other: no alerts

Other effects / acceptance of results:

(Q)SAR model is a valid model for this endpoint and it has been used within its applicability domain

Interpretation of results:

other: non-sensitizer

Conclusions:

DEREK NEXUS version 6.1 did not yield any alerts for skin sensitization of 4-NPP, di-TRIS (CAS 8189-42-4). Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer. 4-NPP, di-TRIS (CAS 8189-42-4) is predicted to be not sensitizing to the skin.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitization of 4-NPP, di-TRIS (CAS 68189-42-4) with the in silico model DEREK NEXUS. In this assessment version 6.1 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 100 alerts for skin sensitization based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitization alerts: The DEREK NEXUS system has been designed for the qualitative prediction of the possible toxicity of chemicals. The predictions made by DEREK NEXUS are intended as an aid to toxicological assessment and, where appropriate, should be used in conjunction with other methods. “No alerts fired” may be extrapolated to a negative prediction.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of 4 -NPP, di-TRIS (CAS 8189-42 -4). Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a nonsensitizer.

Therefore, substance should not be classified according to DEREK NEXUS; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitization for registration under REACH.

The result is adequate to be used in a weight-of-evidence approach together within chemico/in vitro studies to complete the endpoint skin sensitization.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

- A valid DPRA assay was performed according to OECD 442C and GLP principles. The test item was dissolved in ultrapure water at 100 mM and incubated with the cysteine- and lysine-containing synthetic peptides. No precipitate was observed in any of the samples. The mean percent SPCC depletion for the test item was 3.27 % ± 4.68 % and the mean percent SPCL depletion was 0.21 % ± 0.21 %). No co-elution of the test item with SPCC and SPCL has occurred. Therefore, the result is considered to be negative and to show no or minimal reactivity towards the synthetic peptides when using the Cysteine 1:10/Lysine 1:50 prediction model.

- A valid Keratinosens assay was performed according to OECD 442D and GLP principles. The test item was suspended in DMSO at 200mM. From this stock 11 spike solutions were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 – 2000 µM (2-fold dilution series). No precipitate was observed at any dose level tested. Two independent experiments were performed.

The test item showed no toxicity (no IC30 and IC50 value calculated). No biologically relevant, dose-related induction of the luciferase activity was measured in both experiments (No EC_1.5 value calculated). The maximum luciferase activity induction (Imax) was 1.06-fold for experiment 1 and 1.02-fold for experiment 2. 4-NPP, di-TRIS (CAS 68189-42-4) is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed.

- DEREK NEXUS version 6.1 did not yield any alerts for skin sensitization for the test item. 4-NPP, di-TRIS (CAS 68189-42-4) is predicted to be not sensitizing to the skin.

Justification for classification or non-classification

Considering the negative outcome of the DEREK assessment, the DPRA and KeratinoSens assay, 4-NPP, di-TRIS (CAS 68189-42-4) is not considered to be skin sensitizer. Performance of an in vitro assay assessing key event 3 (activation of dendritic cells) is not expected to give additional information as irrespective of the outcome, 4-NPP, di-TRIS is considered to be non skin sensitizer based on the tests currently performed.

Based on the above data, 4-NPP, di-TRIS (CAS 68189-42-4) is not considered to be skin sensitizer. The test item is not classified for skin sensitization according to Regulation 1272/2008 and amendments.