N-ethylpyridazin-4-amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

N-ethylpyridazin-4-amine

EC Number:

846-827-8

Cas Number:

1521274-68-9

Molecular formula:

C6H9N3

IUPAC Name:

N-ethylpyridazin-4-amine

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L2018-006
- Expiration date of the batch: 01 Mar 2020
- Purity: 98.5% (tolerance +/- 1.0 %)
- Physical state / color: solid / white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: Because the test-substance preparation was incubated with the peptide shortly after preparation, no analysis of the test substance in the vehicle was performed.
- Solubility and stability of the test substance in the solvent/vehicle: The stability under storage conditions over the study period was guaranteed.

TEST SUBSTANCE SOLUBILITY
- After stirring and ultrasonication the test substance was soluble (colloid solution) in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 123.16 g/mol and a purity/contents of 98.5%.

OTHER SPECIFICS:
- Identity: confirmed
- Homogeneity: given
- Molecular weight: 123.16 g/mol
- Log KOW: 0.06 (calculated)
- Proposed reaction mechanism for protein binding by OECD toolbox (non-GLP system): The OECD toolbox did not indicate an alert for protein binding for the substance.

In chemico test system

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

- EXPERIMENTAL PROCEDURE
The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

- TEST SUBSTANCE SOLUBILITY
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

- SYNTHETIC PEPTIDES:
-Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
-Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

- CONTROLS
-Negative control (NC): vehicle control = acetonitrile
-Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in acetonitrile
-Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide.

- SELECTION OF CONCENTRATIONS
The test substance was prepared at a 100 mM concentration considering a purity/contents of 98.5% and molecular weight of 123.16 g/mol. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

- PREPARATION OF PEPTIDE STOCK SOLUTIONS
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.

- PREPARATION OF CALIBRATION SAMPLES (see table 1)
The calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer). The analysis of the calibration samples was started before analysis of the test-substance samples.

- TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 123.16 g/mol and a purity/contents of 98.5%. After stirring and ultrasonication the test substance was soluble (colloid solution) in the vehicle.
Vehicle: Acetonitrile
Reason for the vehicle: The test substance was soluble (colloid solution) in acetonitrile.

- PREPARATION OF THE TEST-SUBSTANCE SAMPLES
The samples were prepared in triplicates for each peptide according to the pipetting scheme given in table 2. The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 ± 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

- PREPARATION OF THE VEHICLE CONTROLS
Several vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (deionized water) instead of the test substance:
One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

- PREPARATION OF THE CO-ELUTION CONTROL
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

- MEASUREMENT OF PEPTIDE CONCENTRATIONS
The analyses of the samples were performed via HPLC under the conditions given in table 3.

- DATA EVALUATION
The integrated peak areas were transferred electronically into EXCEL data spreadsheets to carry out the necessary calculations. Some test substances or reaction products may co-eluate with the peptides. In these cases where proper integration and calculation of peptide depletion was not possible, the result for the respective peptide is reported as interference. For evaluation of peptide depletions peak areas at 220 nm are used. When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples (100% ± 10% of the mean of the vehicle controls). However, due to small peak areas calculation of the area ratio may not be possible for all samples (evaluation criteria see tables 4 and 5).

- ACCEPTANCE CRITERIA
- The standard calibration curve should have an r² > 0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- In addition the positive control should cause depletion of both peptides comparable to historic data.

Results and discussion

Positive control results:

Positive control (EGDMA):
Mean depletion (Cysteine-Peptide): 52.76% (+/- 2.72)
Mean depletion (Lysine-Peptide): 9.08% (+/- 0.94)
Mean of both depletions: 30.92%

In vitro / in chemico

Results

Results 1

Parameter:

other: Mean depletion (Cysteine-Peptide) [%]

Value:

1.32

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Parameter:

other: Mean depletion (Lysine-Peptide) [%]

Value:

-0.21

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Parameter:

other: Mean of both depletions [%]

Value:

0.66

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table 6: Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide   mean depletion [%]      SD [%]		Lysine-Peptide   mean depletion [%]        SD [%]		mean of both depletions [%]
test substance	1.32	0.79	-0.21	0.37	0.66
PC:EGDMA in ACN	52.76	2.72	9.08	0.94	30.92

Table 7: Peptide depletion of NC, PC and the test substance for cysteine-peptide.

Reaction with cysteine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean           SD
NC: ACN	-0.13	0.16	-0.03	0.00           0.15
test substance	0.42	1.66	1.89	1.32           0.79
PC:EGDMA in ACN	50.01	52.83	55.45	52.76          2.72

Table 8: Peptide depletion of NC, PC and the test substance for lysine-peptide.

Reaction with lysine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean SD
NC: ACN	-0.05	-0.09	0.14	0.00 0.12
test substance	-0.28	0.19	-0.53	-0.21 0.37
PC:EGDMA in ACN	9.67	9.58	8.00	9.08 0.94

Table 9: Historic control data of negative control / vehicle control (not including present study). Acetonitrile (Historic period: Jan 2017 - Feb 2019)

	C-peptide concentration [mM]	K-peptide concentration [mM]
Min	0.432	0.457
Max	0.564	0.548
Mean	0.492	0.504
SD	0.030	0.015
n	34	33

Table 10: Historic control data of positive control (not including present study). EGDMA, 50 mM in acetonitrile (Historic period: Jan 2017 - Feb 2019)   C-peptide concentration [mM]   C-peptide depletion [%] K-peptide concentration [mM]   K-peptide depletion [%] Min 0.080 46.26 0.395 6.79 Max 0.285 83.79 0.513 19.85 Mean 0.203 58.91 0.445 11.76 SD 0.046 8.17 0.020 2.78 n 32 32 30 30

Applicant's summary and conclusion

Interpretation of results:

other: Non peptide binding/peptide binding

Conclusions:

No predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.
In the present study, the test substance shows minimal or no peptide binding and therefore minimal or no chemical reactivity under the test conditions chosen.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

DPRA:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50

(for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, in order to detect possible interference of the test substance with the peptides, a coelution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm. The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM (colloid solution). The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 1.32%. The mean K-peptide depletion, caused by the test substance was determined to be -0.21%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.66%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model cited in chapter 3.10 it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.