N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-09-07 to 2018-02-28

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))

Version / remarks:

adopted February 04, 2015

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:

N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

EC Number:

205-181-1

EC Name:

N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

Cas Number:

135-16-0

Molecular formula:

C19H23N7O6

IUPAC Name:

(2S)-2-[(4-{[(2-amino-4-oxo-1,4,5,6,7,8-hexahydropteridin-6-yl)methyl]amino}phenyl)formamido]pentanedioic acid

Test material form:

solid

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system)
- Details on study design:
- Pre-experiments: Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following
solvents suitable for the test:
- acetonitrile
- dist. water
- dist. water : acetonitrile 1:1 (v/v),
- isopropanol
- methanol
- 1,4-butanediol
- N,N-dimethylformamide (DMF)
- ethanol
- tert. butanol
The test item was completely soluble in DMF, therefore DMF was chosen as suitable vehicle for the main experiments.
- Preparation of the test item: The test item was freshly prepared immediately prior to use. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.11 was used to correct for the purity of the test item.
- Peptides: 19.90 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.69 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
22.01 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (41.50 mL) to reach a concentration of 0.667 mM.
- Controls:
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides. Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
The incubation scheme is displayed in tabular form under 'any other information on materials and methods'.
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial. After the incubation period the test item was analysed in triplicate for both peptides.
- HPLC: Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred
column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
- Prediction Model: The Prediction Models for both peptides are displayed under 'any other information on material and methods.
- Acceptance criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Results and discussion

Positive control results:

The mean peptide depletion by the positive control was 67.81% Cysteine depletion and 58.82 % Lysine depletion. Results are illustrated under 'any other information on results' in a tabular form.

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: mean percent cysteine depletions

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: highly reactive towards peptide residues

Results 2

Key result

Parameter:

other: mean percent lysine depletions

Value:

1.79

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Values are only considered as estimation due to co-elution, not used for evaluation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: slight precipitations and phase separation in the positive controls were observed. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean peptide concentration for the reference control C was 0.4975 ± 0.0028 mM
- Acceptance criteria met for positive control: yes, mean Peptide concentration for the cysteine peptide was > 60.8 % (67.81 %) and the standard deviation of the positive control was < 14.9% (0.00%), mean Peptide concentration for the lysine peptide was > 40.2% (58.82%) and the standard deviation of the positive control was > 11.6% (0.17 %)
- Acceptance criteria met for variability between replicate measurements: yes, the CV[%] of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0% (0.17 and 0.57 %, respectively)

Any other information on results incl. tables

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1453.8585	0.1612	68.18	67.81	0.64	0.94
	1504.0075	0.1667	67.08
	1453.6510	0.1612	68.18
Test Item	0.0000	0.0013	100.00	100.00	0.00	0.00
	0.0000	0.0013	100.00
	0.0000	0.0013	100.00

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1589.2585	0.1982	59.95	58.82	0.99	1.68
	1662.4030	0.2073	58.11
	1650.5492	0.2058	58.41
Test Item	3944.2044	0.4895	1.62	1.79*	0.17	9.27
	3936.1248	0.4885	1.82
	3930.9854	0.4878	1.95

* A minor/major peak in the co-elution control of the test item was observed at the retention time of the lysine peptide. Therefore, co-elution of test item and peptide occurred and the given peak areas do not properly reflect the amount of lysine peptide present in the test item samples. The values can only be considered as an estimation of the peptide depletion and will not be used for evaluation.

Applicant's summary and conclusion

Interpretation of results:

other: positive results in the direct peptide reactivity assay (DPRA)

Conclusions:

In this study under the given conditions the test item showed high reactivity towards the cysteine peptide. The test item is thus considered to be “positive" in the direct peptide reactivity assay (DPRA). The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.