1,10-Decanediamine, 4-methylbenzenesulfonate (1:2)

Endpoint summary

Description of key information

Result of DPRA according to OECD 422C: Solutions of Clevios K Primer W8 dry were successfully analyzed by the DPRA method in both the Cysteine and Lysine containing synthetic peptides. With no or minimal mean depletion of both peptides (0.866% / 2.46%) in the presence of the test item. Therefore, the DPRA prediction of Clevios K Primer W8 dry is considered negative under the test conditions of this study. However, an underestimated cysteine peptide depletion cannot be fully excluded.

Result of ARE-Nrf2 Luciferase Test according to OECD 422D: The test item, Clevios K Primer W8, gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate peptide buffer and acetonitrile instead of conducting a serial dilution as stated in the OECD 442C Guideline.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Direct Peptide Reactivity Assay (DPRA)

Key result

Run / experiment:

other: Depletion of Cysteine in the presence of Clevios K Primer W8 dry

Parameter:

other: % depletion of peptides

Value:

0.866

Key result

Run / experiment:

other: Depletion of Lysine in the presence of Clevios K Primer W8 dry

Parameter:

other: % depletion of peptides

Value:

2.46

Interpretation of results:

GHS criteria not met

Conclusions:

Solutions of Clevios K Primer W8 dry were successfully analyzed by the DPRA method in both the Cysteine and Lysine containing synthetic peptides. With no or minimal mean depletion of both peptides (0.866% / 2.46%) in the presence of the test item. Therefore, the DPRA prediction of Clevios K Primer W8 dry is considered negative under the test conditions of this study. However, an underestimated cysteine peptide depletion cannot be fully excluded.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Clevios K Primer W8 dry.

The test item was dissolved in methanol when incubated for 24 ± 2 hours at 25 ± 2.5°C. The test item Clevios K Primer W8 dry was analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides.

The DPRA run with the cysteine peptide was repeated once, since the baseline in the chromatograms of the three lowest calibration standards were not flat. In addition, the DPRA run with the lysine peptide was repeated once, since one acceptance criterion was not met.

A visual inspection of the samples immediately upon the preparation showed no precipitations with both peptides. Slight, similar, fluffy precipitations were observed after the incubation time with the cysteine peptide in all samples, with the exception of the four lowest calibration standards and the co-elution samples for the test item and the positive control. The samples were not centrifuged before the HPLC analysis.

The slight, similar, fluffy precipitations might be from the cysteine peptide, possibly from the peptide or from a slight contamination of the peptide, as the precipitations were observed in the calibration standard samples including the two highest concentrations (267 and 534 μM) of the cysteine peptide and in all other samples including a cysteine peptide concentration of 500 μM. In the OECD 442C guideline it is stated that, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Since the r2 of the standard calibration curve met the acceptance criterion, the cysteine peptide depletion of the positive control was within our historical data and the reference controls met the acceptance criteria, the peptide depletion results – negative result - of the test item samples are considered acceptable.

For verification of the cysteine peptide analysis, one additional HPLC analysis with the cysteine peptide of another peptide provider (RS synthesis) was conducted for verification. In this verification experiment no precipitations were observed immediately upon the preparation of the samples. But slight fluffy precipitations were observed after the incubation time with the cysteine peptide in all samples, with the exception of the four lowest calibration standards and the co-elution samples for the test item and the positive control (as observed in the previous HPLC analysis with the cysteine peptide from JPT). Since the result of the verification experiment was similar to the result of the previous cysteine peptide analysis, the DPRA prediction of the test item is considered negative under the test conditions of this study. However, an underestimated cysteine peptide depletion cannot be fully excluded.

In addition, milky precipitations were observed after the incubation time with the lysine peptide in the positive control samples and in the co-elution samples of the positive control. Since the mean depletion of the positive control was within the range of the acceptance criteria, this result was considered acceptable.

With an overall depletion value of 0.866% in the first analysis and 2.46% in the verification experiment (applying the depletion model for cysteine and lysine), Clevios K Primer W8 dry is placed in the reactivity class of “no or minimal” and the DPRA prediction is considered negative.

In conclusion, solutions of Clevios K Primer W8 dry were successfully analyzed by the DPRA method in both the Cysteine and Lysine containing synthetic peptides. With no or minimal mean depletion of both peptides (0.866% / 2.46%) in the presence of the test item. Therefore, the DPRA prediction of Clevios K Primer W8 dry is considered negative under the test conditions of this study. However, an underestimated cysteine peptide depletion cannot be fully excluded.

Reference 2

Endpoint:

skin sensitisation: in vitro

Remarks:

ARE-Nrf2 Luciferase Test (KeratinoSens™)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 1

Parameter:

other: Imax

Value:

2.06

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 1

Parameter:

other: μg/mL EC1.5

Value:

248.91

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 1

Parameter:

other: μg/mL IC30

Value:

196.45

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 1

Parameter:

other: μg/mL IC50

Value:

272.36

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 1

Parameter:

other: % cellular viability at the EC1.5 determining concentration

Value:

70

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 2

Parameter:

other: Imax

Value:

2.1

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 2

Parameter:

other: μg/mL EC1.5

Value:

279.73

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 2

Parameter:

other: μg/mL IC30

Value:

201.57

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 2

Parameter:

other: μg/mL IC50

Value:

290.6

Key result

Run / experiment:

other: Results for Clevios K Primer W8 - Test 2

Parameter:

other: % cellular vialbility at the EC1.5 determining concentration

Value:

70

Other effects / acceptance of results:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Clevios K Primer W8, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for Clevios K Primer W8 was 2.06 in test 1 and 2.10 in test 2. The Imax for both tests was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 248.91 μg/mL and 279.73 μg/mL for tests 1 and 2, respectively. As the EC1.5 was >200 mg/mL, the KeratinoSens™ prediction was negative. The IC30 value was 196.45 μg/mL in test 1 and 201.57 μg/mL in test 2 and the IC50 values were 272.36 μg/mL and 290.60 μg/mL in tests 1 and 2, respectively. Graphs showed no overall dose-response for luciferase induction.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that the test item, Clevios K Primer W8, gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that the test item, Clevios K Primer W8, gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Clevios K Primer W8, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for Clevios K Primer W8 was 2.06 in test 1 and 2.10 in test 2. The Imax for both tests was >1.5 fold and statistically significant compared to the DMSO control. The EC1.5 was 248.91 μg/mL and 279.73 μg/mL for tests 1 and 2, respectively. As the EC1.5 was >200 mg/mL, the KeratinoSens™ prediction was negative. The IC30 value was 196.45 μg/mL in test 1 and 201.57 μg/mL in test 2 and the IC50 values were 272.36 μg/mL and 290.60 μg/mL in tests 1 and 2, respectively. Graphs showed no overall dose-response for luciferase induction.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that the test item, Clevios K Primer W8, gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)