Monalazone disodium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 Nov 2016 - 27 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

no

Remarks:

The study has been conducted for US authorities originally but used here as WoE.

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

Monalazone disodium

EC Number:

262-810-2

EC Name:

Monalazone disodium

Cas Number:

61477-95-0

Molecular formula:

C7H4ClNO4S.2Na

IUPAC Name:

disodium 4-[(chloroazanidyl)sulfonyl]benzoate

Specific details on test material used for the study:

Lot#: 52716BC13
Storage: RT
Purity: Mixture with 20% active ingredient (monalazone disodium)

In chemico test system

Details on the study design:

Test & Reference Articles
Test article was received by Cyprotex lab staff and stored at room temperature. Urine was received from LeeBio Solutions and stored at 2-8°C. Positive control, 2,3-butanedione (CAS 431-03-8), was prepared at 100 mM in acetonitrile (CAS 75-05-8, Lot 150041).
Compound Name (purpose) Lot # Storage MW Purity/comments
2,3-butanedione (positive control) MKBB5095V 2 – 8°C 86.09 97% purity
Test Item (Monalazone Disodium) 52716BC13 RT NA Mixture with 20% active ingredient. See section 3c for specific formulations of test article
Fresh Human Urine (reference for urine reacted test article) W236942 (Lysine)W236942, W237946, 12A2351(Cysteine) 2 – 8°C NA Complex mixture
RT = Room Temperature

Justification of Test System
Direct Peptide Reactivity Assay: DPRA was developed by Frank Gerberick and colleagues (2004) and was further refined in 2007 (Gerberick et al., 2007), and a full OECD guideline for the assay was released in February of 2015. In this assay, the test article was incubated concurrently in two separate buffers, one with cysteine (Ac-RFAACAA-COOH) and one with lysine (Ac-RFAAKAA-COOH). Reactive chemicals bind one or both of the peptides thereby reducing their free concentration levels. The disappearance of each peptide is measured by HPLC/UV. This method does not require any biological material such as enzymes in order for this reaction to take place. It is important to note that the cysteine peptide captures soft electrophiles, while the lysine peptide captures hard electrophiles. This makes the DPRA assay a good choice to screen for reactive chemicals which are associated with allergic contact dermatitis.

Preparation of Samples
Test Article Stock: Urine was added to reactions undiluted at the same volume as the 2,3-butanedione reference chemical. In all testing conditions test article formulations were added at the same volume as the 2,3-butanedione reference chemical. The test chemical was tested in four different ways:

1. Monalazone disodium (20%)
2. A theoretical 100 mM (3.33%) active ingredient preparation prepared by mixing 4.16g 20% monalazone with ultrapure water 20.84g water
3. Monalazone disodium (20%) diluted to in use concentrations in water prepared by mixing 0.375g 20% monalazone with 24.625g ultrapure water.
4. Monalazone disodium (20%) diluted to in use concentrations in urine prepared by mixing 0.375 g 20% monalazone with 24.625 g urine.

Reference materials: 2,3-butanedione was prepared in acetonitrile (Optima LC/MS, 99.9%, Fisher, Waltham, MA, Lot No. 160783, and CAS 75-05-8) to yield a final concentration of 100 mM.

Peptides: A 0.667 mM stock solution of the lysine peptide was prepared by diluting the peptide with Lysine Reaction buffer, and a 0.667 mM stock solution of cysteine peptide was prepared by diluting the peptide in Cysteine Peptide Reaction buffer. Unprepared peptides were stored at approximately -80°C desiccated. Peptides were from LifeTein LLC (South Plainfield, NJ).
Peptide Supplier Lot Number Weight (mg) Volume (mL) Concentration
Cysteine Lifetein LT160926-LT180433 19.2 38.3 0.667mM
Lysine Lifetein LT151110-LT107617 13.2 25.5 0.667mM

Evaluation of Test Article in the Assay
DPRA: DPRA was run according to Cyprotex SOP-2078. The cysteine peptide was prepared at 0.667 mM in Cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer as outlined in OECD 442c. The reaction mix for cysteine peptide had a 1:10 test peptide to reference article ratio (0.5 mM cysteine to 5 mM reference article). The reaction mix for lysine peptide had a 1:50 peptide to reference article ratio (0.5 mM lysine to 25 mM reference article). Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile or water containing no reference or test article.
Reference control reactions were prepared by mixing 2.5 mL of acetonitrile with 7.5 mL of the respective buffer. Aliquots of 1 mL were added to 9 glass vials for each peptide and placed at the beginning middle and end of the samples as shown in Annex 1. These reactions were used to ensure the consistency of peptide detection during the run.
A standard curve was prepared for both peptides by adding 400 µL of acetonitrile to 1600 µL of 0.667 mM peptide to make a 0.534 mM standard. This 0.534 mM standard was serial diluted in 20% acetonitrile/buffer to make a 6 point standard curve. A zero peptide standard was also included in the standard curve.
After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. The samples were transferred to WMed Innovation Center for peptide assessment. The Lysine reactions were analyzed via HPLC with gradient elution and UV detections at 220 nm using a Waters Alliance 2795 HPLC equipped with a 2996 Photodiode Array. Prior to sample analysis the suitability of the HPLC/UV system was verified by running the standard curve and a triplicate set of reference controls (see Annex 1). Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Sample analysis was initiated within 24 ±2 hours of the reaction start. The results were acquired with the MassLynx data system and quantified via the QuanLynx application. Cysteine peptide reactions were analyzed via HPLC with gradient elution and UV detections at 220 nm using a Agilent 1100 HPLC equipped with a diode array. Prior to sample analysis the suitability of the HPLC/UV system was verified by running the standard curve and a triplicate set of reference controls (see Annex 1). Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Sample analysis was initiated within 24 ±2 hours of the reaction start. The results were acquired with the Agilent ChemStation for LC 3D systems. Samples were injected once.
After determination of peptide remaining in the analysis, percent depletion relative to vehicle controls was calculated relative to no test article (vehicle control) samples. Peptide reactivity was reported as percent depletion and was calculated using the following formula:
% Depletion = (1-(test compound area/ vehicle control area)) x 100

Results and discussion

Positive control results:

The mean Percent Lys Depletion for the positive control 2,3-butanedione was 22.4%.
The mean Percent Cys Depletion for the positive control cinnamic aldehyde was 81.7%.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: % Lys depletion

Remarks:

20% monalazone disodium

Value:

94.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: 1

Parameter:

other: % Cys depletion

Remarks:

20% monalazone disodium

Value:

45.2

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Results 3

Key result

Run / experiment:

other: 1

Parameter:

other: % Depletion DPRA

Value:

69.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Other effects / acceptance of results:

Depletion of 2,3-butanedione fell into the expected in range for the assay according to the OECD guideline. The standard deviations for 2,3-butanedione and acetonitrile in the cysteine reactions were outside the expected ranges outlined in the OECD guideline and our historic observations. This is unsurprising given the behavior of the test material.

System suitability was shown by examining the r2 value for the standard curves and the average, standard deviation, and % coefficient of variation of the reference control samples. The calibration curves for both peptides were shown to have r2 values of greater than 0.99. The cysteine standard curve had an r2 value of 0.999585, while the lysine standard curve had an r2 value of 0.995273.

Reference controls containing only peptide, acetonitrile and buffer were run at the beginning, middle, and end of each peptide series to show stability. Cysteine reference controls run after the 20% monalazone samples showed a marked reduction in the amount of cysteine peptide present. Running the samples separately revealed that the reduction was due to interference from the test article.

Each test and reference article was measured in triplicate in both peptides.

Performance guidelines in OECD 442c state that Standard Deviation of the test chemical replicates should be less than 14.9% for the cysteine peptide depletion and less than 11.6% for the lysine peptide depletion. Because of the test material retention on the column, these criteria were not met for all the test and reference articles. The client test article and urine performed acceptably in the lysine reactions. Peptide depletion was noted and was proportional to the amount of test chemical added. Slight precipitation was observed in the in use urine reactions, but depletion was observed never the less.
The performance with the cysteine peptide was fairly clear that the test material is highly reactive with the cysteine peptide. However, because the analytical methodology was not ideal for the test article the study should be considered inconclusive.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The study should be considered inconclusive because the analytical methodology was not ideal for Monalazone disodium.

Executive summary:

The purpose of this study was to screen 20% Monalazone Disodium, for its potential to act as chemical sensitizers using the Direct Peptide Reactivity Assay (DPRA), a test used to assay reactivity of test articles with small peptides.

The test material was formulated in four different ways to analyze its properties at both bulk and in use concentrations.  A summary of the results for the test article formulations and control article is provided in table below. 

Material	%Lys Dep	%Cys Dep*	%Dep DPRA	Reactivity Class	Sensitizer
2,3-butanedione	22.4	81.7	52.0	High	Inconclusive
Urine	16.8	90.5	53.6	High	Inconclusive
20% monalazone disodium	94.4	45.2	69.8	High	Inconclusive
100 mM monalazone disodium (theoretical)	68.4	12.9	40.7	Moderate	Inconclusive
In use water 20% monalazone disodium	17.0	92.9	55.0	High	Inconclusive
In use urine 20% monalazone disodium	17.2	99.9	58.5	High	Inconclusive

*depletion calculated relative to reference controls run apart from test articles

 

An overall confounding factor for this study was the behavior of the test material in cysteine reactions during analysis. The reactions were performed three times in attempts to determine exactly what was occurring in the test system. It was observed that after running any samples containing client test material, subsequent reference reactions (containing vehicle, peptide and buffer) showed a marked decrease in peptide level compared to the expected 500 μM amount. The reference reactions were run in sets of three, and it was observed that the amount of peptide in the first replicate was very low, the next slightly higher and the final almost to the expected 500 μM level. An attempt was made to move the samples that presumably caused this issue to the end of the run; however, the issue persisted and it seems likely that any test item on the column adversely affects the downstream samples. This makes the results for the cysteine reactions highly questionable. Since there is no model using only the lysine peptide, an attempt was made to calculate the Cysteine depletion by comparing the sample data to reference controls which were run apart from the test materials. These controls showed the expected level of peptide when run prior to the test material samples, indicating they were prepared correctly but affected by the test materials when run with them. 

 

Depletion of 2,3-butanedione fell into the expected in range for the assay according to the OECD guideline. The standard deviations for 2,3-butanedione and acetonitrile in the cysteine reactions were outside the expected ranges outlined in the OECD guideline and our historic observations. This is unsurprising given the behavior of the test material. 

 

The client test article and urine performed acceptably in the lysine reactions. Peptide depletion was noted and was proportional to the amount of test chemical added. Slight precipitation was observed in the in use urine reactions, but depletion was observed never the less.  The performance with the cysteine peptide is outlined above and it was fairly clear that the test material is highly reactive with the cysteine peptide. However, because the analytical methodology was not ideal for the test chemical, the study should be considered inconclusive.