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EC number: 213-037-4 | CAS number: 918-04-7
Table 6: Peptide depletion for C-peptide
Reaction with cysteine- peptide
peptide depletion [%]
PC: EGDMA in H2O
Table 7: Peptide depletion for K-peptide
Reaction with lysine-peptide
Table 8: Mean peptide depletions
mean of both depletions[%]
Table 9a: Historic control data of vehicle control (de-ionized water) (not including present study)
Table 9b: Historic control data of positive control (EGDMA, 50 mM in de-ionized water) (not including present study)
C-peptide depletion [%]
K-peptide depletion [%]
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 UVdetection at 220 nm.
The test substance was dissolved at ca. 100 mM concentration in de-ionized water (At the time of the conduct of the DPRA preliminary information on the content of the test substance was available and used for calculation of 100 mM concentration (53.9 g/ 100 g). However, the final content of the test substance is reported to be 50.7 g /100 g. Hence, considering the final purity, a concentration of ca. 94 mM was used as stock concentration of the DPRA). 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, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.
The following results were obtained in the DPRA:
The test substance was dissolved in de-ionized water at a concentration of ca. 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 -7.41%. The mean K-peptide depletion, caused by the test substance was determined to be -0.05%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.
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.
However, it should be noted, that due to the lower concentration of the stock concentration used, the result could be under-predictive.
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