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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Mar - Apr 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 Rheinland-Pfalz
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
(1,2-dimethylpropyl)-5-methyl-pyrazole-4-carboxylic acid
EC Number:
846-828-3
Cas Number:
1872341-39-3
Molecular formula:
C10H16N2O2
IUPAC Name:
(1,2-dimethylpropyl)-5-methyl-pyrazole-4-carboxylic acid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- Physical state / color: solid / yellowish
- Molecular weight: 196.2 g/mol
- Log Kow: 2.85 (calculated)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 196.2 g/mol and a purity / contents of 99.6 %. After stirring and ultra-sonication the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution

In chemico test system

Details on the study design:
Skin sensitisation (In chemico test system) - Details on study design:
The test substance was dissolved in a suitable vehicle. Per run 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.

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 THE TEST SUBSTANCE SAMPLES
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 HPLC 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 (acetonitrile) 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.

Results and discussion

In vitro / in chemico

Results
Run / experiment:
other: cysteine-peptide
Parameter:
other: mean peptide depletion [%]
Value:
10.52
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 5: Summary of mean peptide depletions of Cysteine and Lysine

 

Cysteine-peptide

Lysine-peptide

1sttest run

Lysine-peptide

2ndtest run

Lysine-peptide

3rdtest run

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Test substance

10.52

2.13

0.66

0.41

interference

interference

PC: EGDMA in ACN

52.76

2.72

interference

9.08

0.94

9.70

0.75

Table 6: Historic control data of negative control / vehicle control (not including present study), 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 7: Historic control data of positive control (not including present study), 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

30

Applicant's summary and conclusion

Interpretation of results:
other: no peptide binding potential
Conclusions:
Based on the observed results and applying the cysteine 1:10 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.
Executive summary:

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. For the C-containing peptide, one test run was performed. For the K-containing peptide, three test runs were performed. Per test run 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. 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 C-containg peptide was present. For the K-peptide reaction co-elution was noticed using two different analysis methods. The mean C-peptide depletion, caused by the test substance was determined to be 10.52 %. The mean K-peptide depletion, caused by the test substance was determined to be 0.66 % in a first test run. However, although no co-elution was present, this reaction is considered to be formally invalid, as co-elution was noticed in the positive control due to insufficient separation of the analytic column. In two further test runs, using the standard and alternative analysis method and a new analytic column, co-elution was noticed in the samples of the test substance. Due to the interference observed in the K-peptide samples, calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

Based on the observed results and applying the cysteine 1:10 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.