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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:
May - Jun 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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: solid, white
- Molecular weight: 261.794 g/mol
- Log Kow: - 0.77 (information provided by the sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient; under N2
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in the vehicle.

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 261.794 g/mol and a purity / contents of 91.1 %. After short stirring 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 study design:
Skin sensitisation (In chemico test system) - Details on study design:
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.

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 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 (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

Resultsopen allclose all
Parameter:
other: Mean peptide depletion [%]
Run / experiment:
mean of both depletions
Value:
0.04
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: C-peptide depletion [%]
Run / experiment:
mean
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
negative results were set to "0"
Parameter:
other: L-peptide depletion [%]
Run / experiment:
mean
Value:
0.08
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Table 5: 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

- 0.05

0.17

- 0.11

0.00

0.15

Test substance

- 0.29

- 0.34

0.28

- 0.12

0.34

PC: EGDMA in ACN

50.31

51.92

53.42

51.88

1.56

Table 6: Peptide depletion of NC, PC and the test substance for lysine-peptide

Reaction with cysteine-peptide

Peptide depletion [%]

Sample 1

Sample 2

Sample 3

Mean

SD

NC: ACN

- 0.19

- 0.62

0.81

0.00

0.74

Test substance

0.00

0.15

0.08

0.08

0.07

PC: EGDMA in ACN

11.47

12.92

13.04

12.48

0.87

Table 7: Mean peptide depletions of Cysteine, Lysine and both peptides

 

Cysteine-Peptide

Lysine-Peptide

Mean of both depletions [%]

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Test substance

- 0.12

0.34

0.08

0.07

0.04

PC: EGDMA in ACN

51.88

1.56

12.48

0.87

32.18

Applicant's summary and conclusion

Conclusions:
Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance was 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. 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 peptides was present.

The mean C-peptide depletion, caused by the test substance was determined to be - 0.12 %.

The mean K-peptide depletion, caused by the test substance was determined to be 0.08 %.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.04 %.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.