5-amino-2-nitrobenzoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 - 19 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

18 June 2019

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

Constituent 1

Reference substance name:

5-amino-2-nitrobenzoic acid

EC Number:

236-283-4

EC Name:

5-amino-2-nitrobenzoic acid

Cas Number:

13280-60-9

Molecular formula:

C7H6N2O4

IUPAC Name:

5-amino-2-nitrobenzoic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

Unknown impurities.

Molecular formula:

Not available as unknown impurities.

IUPAC Name:

Unknown impurities.

Test material form:

solid

Details on test material:

Batch / Lot No. 10044431

Specific details on test material used for the study:

Batch-No.: 10044431
Storage: room temperature

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 27 hours.

Steps of the DPRA Method done in chronological order
- Solubility assessment of the test chemical – 1:1 acetonitrile: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 acetonitrile50 µL 100mM test item solution (or solvent for the reference controls A,B,C or 100
mM 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 con
trol solution for the positive control)

The vials were capped, vortexed to mix and placed to the HPLC autosampler for 24 ± 2 h incubation at 25 ± 2.5 °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 controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended.


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 did not dissolve the test item, it sank to the bottom of the test tube even after vortexing and no total dissolution could be observed.
The test item was also tried to be dissolved in ultrapure water but the test item and solvent separated intro two phases. However the 1:1 mixture of acetonitrile:ultrapure water dissolved the test item completely, a homogenous and transparent solution was formed after vortexing at 100 mM. 2-propanol did not dissolve the test item even after vortexing, while dissolving in acetone resulted in a clear solution. Trying to dissolve the test item with the 1:1 mixture of acetone:acetonitrile resulted in an unclear solution. Dissolution in DMSO was also tried before diluting the gained solution with acetonitrile in the guideline recommended ratios (first 100 µL DMSO then 900 µL acetonitrile or first 500 µL DMSO then 500 µL acetonitrile).
The solubility test resulted in four formulation that complies with the requirements of the guideline at the concentration of 100 mM in case of the following solvents: 1:1 mixture of acetonitrile:ultrapure water; acetone; DMSO (100 µL) + acetonitrile (900 µL); DMSO (500 µL) + acetonitrile (500 µL).
First, the behavior of the formulation of the test item with the 1:1 mixture of acetonitrile: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, transparent solutions were obtained. Since this solvent is the preferred vehicle in the order of solvents listed in the guideline OECD TG 442C Guideline for the testing of Chemicals - In Chemico Skin Sensitization: Direct Peptide Reactivity Assay (DPRA) (18 June 2019) and the formulation complied with all obligations of the test guideline, no more formulations were checked but 1:1 mixture of acetonitrile:ultrapure water was chosen.
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.

Synthetic Peptides
Cysteine peptide
Name: Cysteine peptide
Batch no: 111016HS-MHeW0819
Storage: at -20°C or below
Purity: 91.16 %
Molecular weight: 750.88 g/mol
Sequence: Ac-RFAACAA-OH
Expiry date: February 16, 2020

Lysine peptide
Name: Lysine peptide
Batch no: 020517HS_MHeW0819
Storage: at -20°C or below
Purity: 95.73 %
Molecular weight: 775.91 g/mol
Sequence: Ac-RFAAKAA-OH
Expiry date: February 20, 2020

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

Percent peptide depletion
The concentration of the peptide was determined in each reaction 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 rpocess 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”.
"Prediciton model to be chosen":
no co-elution: Cysteine 1:10 / Lysine 1:10 prediciton model
co-elution with only cysteine or cysteine and lysine peptides: inconclusive
co-elution with only lysine: Cysteine 1:10 prediciton 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, 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 77.04 % ± 2.37 % and a mean lysine peptide depletion value of 53.03% ± 0.92 %.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

42.87 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.92 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no relevant increase

Outcome of the prediction model:

low reactivity [in chemico]

Other effects / acceptance of results:

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

Co-elution
The test chemical did not absorb at 220 nm significantly (> 10 % compared to the respective reference control) when tested with the cysteine and lysine peptides. Therefore no co-elution was observed with either of the peptides.
The range of retention time for cysteine peptide was between 8.124 and 8.514 and the range of retention time for lysine peptide was between 6.137 and 6.294.

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 0.48 mM for the cysteine and 0.51 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 analysed by linear regression.
Means of the peak areas versus the concentrations of both peptides showed good linearity with r2 = 0.9977 for the cysteine peptide and r2 = 0.9999 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 much smaller than the acceptable 15 % for both peptides, since it was 2.8 % for cysteine and 0.1 % for lysine peptides. 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 peptide concentration of the reference control C (solvent – ultrapure 1:1 water:acetonitrile – upw:acn) replicates was 0.45 mM, and the mean lysine peptide concentration of the reference control C (solvent – ultrapure 1:1 water:acetonitrile – upw:acn) replicates was 0.50 mM, which were within the acceptable 0.50 ± 0.05 mM range.
The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 77.04 % ± 2.37 % and a mean lysine peptide depletion value of 53.03% ± 0.92 %.
The percent cysteine peptide depletion with the test item was 42.87 % ± 9.08 % while the percent lysine peptide depletion with the test item was 0.92 % ± 0.34 %.
The mean peptide depletion value for the positive control was 65.04 % and mean peptide depletion value for the test chemical was 21.90 %

Assigning the test chemical to a reactivity class and category
The average percent peptide depletion was calculated for the test item. Since no co-elution was observed, applying the cysteine 1:10 / lysine 1:50 prediction model, the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. The mean percent peptide depletion value was 21.90 %. Thus, the test item is considered to be positive in the DPRA and classified in the low reactivity class when using the cysteine 1:10 / lysine 1:50 prediction model.

Protocol Deviations
No deviations.

Demonstration of technical proficiency
Prior to routine use of the method, TOXI-COOP ZRT. demonstrated technical proficiency in a separate study (Study number.: 392-442-2996) by correctly obtaining the expected DPRA prediction for 10 proficiency substances as recommended in the OECD TG 442C guideline.

Applicant's summary and conclusion

Interpretation of results:

other: potential skin sensitizer

Conclusions:

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “5-Amino-2-Nitrobenzoic Acid (CAS 13280-60-9)” was concluded to be positive and to show low reactivity towards the synthetic peptides thus is 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 “5-Amino-2-Nitrobenzoic Acid (CAS 13280-60-9)” 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 and the two valid runs were included in the prediction model for the classification of the test item.

Peptide depletion resulted from the positive control cinnamaldehyde was 77.04 % ± 2.37 % with cysteine peptide and 53.03 % ± 0.92 % 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 (0.45 – 0.48 mM) and lysine peptides (0.50 mM – 0.51 mM) and the CV % for the for the nine reference controls B and C in acetonitrile were 2.8 % and 0.1 % percentages for the cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay.

The percent cysteine peptide depletion value of the test item was 42.87 % ± 9.08 % while the percent lysine peptide depletion was 0.92 % ± 0.34 %.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 21.90 %, which exceeded the 6.38 % threshold of the applicable prediction model and fell into the low reactivity class.

Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item “5-Amino-2-Nitrobenzoic Acid (CAS 13280-60-9)” was concluded to be positive and to show low reactivity towards the synthetic peptides thus is a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.