Fenuron

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In vitro studies need to be performed as first step.

Test material

Test material information

1

Reference substance name:

Fenuron

EC Number:

202-941-4

EC Name:

Fenuron

Cas Number:

101-42-8

Molecular formula:

C9H12N2O

IUPAC Name:

fenuron

Test material form:

other: white to off-white powder

Details on test material:

- Particle size distribution: 100 % particle size < 12 µm (method: Laser Diffraction; Certificate of Analysis)
Particle size parameter determined with a Malvern Mastersizer 2000 (Non-GLP determination)
D10% = 1.18 µm
D50% = 7.72 µm
D90% = 16.00 µm
D: Diameter; 10, 50, 90: percentage cumulative
- Mass median aerodynamic diameter (MMAD): 1.595 µm
- Geometric standard deviation (GSD): calculated as 2.09

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0010416
- Expiration date of the lot/batch: April 2019
- Purity test date: 05 october 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At +10 to +25°C, in a tightly closed container in a dry, cool and well-ventilated place, avoid exposure to sunlight and moisture

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solubility of the test item in an appropriate solvent was assessed before performing the assay. 49.76 mg IsoQure UR 300 were dissolved in 3 mL acetonitrile immediately before testing to prepare a 100 mM solution. A factor of 1.01 was used to correct for the purity of the test item. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.

OTHER SPECIFICS:Trade name: IsoQure UR 300

In chemico test system

Details on the study design:

PROCEDURE
-Preparation of the cysteine or lysine-containing peptides:
Stock solutions of cysteine (Ac-RFAACAA-COOH) and lysine (Ac-RFAAKAA-COOH) containing synthetic peptides of purity higher than 95% were freshly prepared just before their incubation with the test item. The final concentration of the cysteine peptide was 0.667 mM in pH 7.5 phosphate buffer, whereas the final concentration of the lysine peptide was 0.667 mM in pH 10.2 ammonium acetate buffer.
-Preparation of the test item:
Solubility of the test item in an appropriate solvent was assessed before performing the assay. 49.76 mg IsoQure UR 300 were dissolved in 3 mL acetonitrile immediately before testing to prepare a 100 mM solution. A factor of 1.01 was used to correct for the purity of the test item. The test item solution was then tested as such without any further dilution by incubating at 1:10 or 1:50 ratio with the cysteine peptides and lysine peptides, respectively.
-Positive control, reference controls and coelution control:
Cinnamic aldehyde (CAS no. 14371-10-9) was used as positive control (PC) at a concentration of 100 mM in acetonitrile. In addition reference controls (i.e. samples containing only the peptide and added acetonitrile) were also included in the HPLC run sequence and these were used to verify the HPLC system suitability prior to the analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. In addition a coelution control constituted by the test item alone for the test item analysed was included in the run sequence to detect possible coelution of the test item with either the lysine or the cysteine peptide.
-Incubation of the test item with the cysteine and lysine peptide solutions:
Cysteine and lysine peptide solutions were incubated in glass autosampler vials with the test item at 1:10 and 1:50 ratio, respectively. The reaction solution was left in the dark at 25 ± 2.5°C for 24 ± 2 hours before running the HPLC analysis. The test item assay was analyzed in triplicate for both peptides. Samples were visually inspected prior to HPLC analysis. If a precipitate would be observed immediately upon addition of the test item solution to the peptide solution, due to low aqueous solubility of the test item, in this case one cannot be sure how much test item remained in the solution to react with the peptide. Therefore, in such a case, a positive result could still be used, but a negative result is uncertain and would be interpreted with due care. No precipitate or phase separation was observed.
-Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both the cysteine and the lysine peptides. Peptide standards were prepared in a solution of 20% acetonitrile : buffer using 100 mM sodium phosphate buffer (pH 7.5) for the cysteine peptide and 100 mM ammonium acetate buffer (pH 10.2) for the lysine peptide. Using serial dilution standards of the peptide stock solution (nominal concentrations: 0.666 mM of cysteine peptide in sodium phosphate or 0.666 mM lysine peptide in ammonium acetate), 6 calibration standards were prepared to cover the range from 0.534 to 0.0167 mM. A blank of the dilution buffer was also included in the standard calibration curve. Suitable calibration curves should have an r2 > 0.99.
-HPLC preparation and analysis:
If a test item promotes the oxidation of the cysteine peptide, the peak of the dimerised cysteine peptide would have been visually monitored. If dimerisation appears to have occurred, this would have been noted as percent peptide depletion which would have been over-estimated leading to false positive predictions and/or assignment to a higher reactivity class. No dimerisation of the cysteine peptide occurred. HPLC analysis for the cysteine and lysine peptides was performed on one day. All test item solutions were freshly prepared for both assays on one day. The analysis was timed to assure that the injection of the first sample (reference control C) starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC run sequences were set up in order to keep the HPLC analysis time to less than 30 hours.
- Evaluation
The concentrations of cysteine or lysine peptide were photometrically determined at 220 nm in each sample by measuring the peak area (area under the curve, AUC) of the appropriate peaks and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
The percent peptide depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference control C.
- Acceptance criteria
The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.
The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.
- Prediction model
The mean percent cysteine and percent lysine depletion value was calculated for each test item. Negative depletion was considered as “0” when calculating the mean. By using the cysteine 1:10/lysine 1:50 prediction model , the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of an Integrated Approach to Testing and Assessment (IATA). Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate and high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA.
No coelution was observed.
A single HPLC analysis for both the cysteine and the lysine peptide would be sufficient for a test item when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. If situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine 1:10/lysine 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run would be considered, as well as a third one in case of discordant results between the first two runs.

Results and discussion

Positive control results:

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.81% for cysteine and 51.15% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

In vitro / in chemico

Results

Run / experiment:

other: Cysteina+ Lysine

Parameter:

other: % depletion

Value:

2.58

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

Reactivity Class (Cysteine+ Lysine) = Negative / Reactivity Class (Cysteine only) = Negative

Other effects / acceptance of results:

Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and acetonitrile were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate.
No precipitate in the reaction mixture at the end of the incubation time and no coelution were observed.
IsoQure UR 300-treated samples revealed a cysteine peptide depletion of 3.02% and a lysine peptide depletion of 2.14% (mean peptide depletion of 2.580%) and, hence, were well below 6.38%. IsoQure UR 300 is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).
Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.81% for cysteine and 51.15% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The linearity of the standard calibration curve was r2 = 1.0000 for cysteine peptide and r2 = 0.9999 for lysine peptide. Hence the requirement of r2 > 0.99 was met.
The mean peptide concentrations of reference control A were 0.508 or 0.494 mM cysteine or lysine peptide, respectively and, hence well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C was <15.0%.

The mean peptide concentrations of reference control A were 0.508 or 0.494 mM cysteine or lysine peptide, respectively and, hence well within the accepted range of 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C was <15.0%.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

IsoQure UR 300 revealed a mean cysteine and lysine peptide depletion of 2.580% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Executive summary:

The purpose of this study was to determine the sensitising potential of IsoQure UR 300 in a Direct Peptide Reactivity Assay (DPRA). The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers. The test item was dissolved at a concentration of 100 mM in acetonitrile. Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and acetonitrile were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate. IsoQure UR 300-treated samples revealed a cysteine peptide depletion of 3.02% and a lysine peptide depletion of 2.14% (mean peptide depletion of 2.580%) and, hence, were well below 6.38%. IsoQure UR 300 is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA). Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 70.81% for cysteine and 51.15% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The acceptance criteria of validity were fulfilled in this test.