Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-14 to 2019-01-18
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04 February 2015
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
Version / remarks:
January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use

Test material

Constituent 1
Test material form:
liquid

In chemico test system

Details on the study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Experimental Procedure
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.
Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the HPLC procedure.

Results and discussion

Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion
Value:
1.55
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value in %
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion
Value:
0.76
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value in %
Other effects / acceptance of results:
Acceptance Criteria
Cysteine Peptide Run
- coefficient of determination R² > 0.99: 0.9999 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM: 0.5097 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM: 0.5049 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM: 0.4945 pass
- CV of the peak area of RC B < 15%: 0.81 pass
- CV of the peak area of RC C (PC) < 15%: 0.34 pass
- CV of the peak area of RC C (TI) < 15%: 1.97 pass
- mean peptide depletion of the PC 60.8% < x < 100%: 69.38 pass
- SD of peptide depletion of the PC replicates < 14.9%: 0.58 pass
- SD of peptide depletion of the TI replicates < 14.9%: 1.40 pass

Lysine Peptide Run
- coefficient of determination R² > 0.99: 1.0000 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM: 0.5014 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM: 0.5001 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM: 0.5067 pass
- CV of the peak area of RC B < 15%: 0.84 pass
- CV of the peak area of RC C (PC) < 15%: 0.26 pass
- CV of the peak area of RC C (TI) < 15%: 0.43 pass
- mean peptide depletion of the PC 40.2% < x < 69.0%: 66.34 pass
- SD of peptide depletion of the PC replicates < 11.6%: 0.83 pass
- SD of peptide depletion of the TI replicates < 11.6%: 1.20 pass

Both peptide runs and the test item results met the acceptance criteria of the test.

Any other information on results incl. tables

In the present study the test item was dissolved in isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 395.66 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cisopropanol).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (1.15%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.

The controls confirmed the validity of the study for both, the cysteine and lysine run.

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

16.4390

0.5340

14.6340

0.5340

STD2

8.2610

0.2670

7.3130

0.2670

STD3

4.0740

0.1335

3.6450

0.1335

STD4

1.9450

0.0667

1.8080

0.0667

STD5

0.9340

0.0334

0.9000

0.0334

STD6

0.4240

0.0167

0.4440

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.7360

0.1551

69.56

69.38

0.58

0.84

4.8660

0.1593

68.73

4.6910

0.1537

69.85

Test Item

14.8270

0.4812

2.72

1.55

1.40

90.32

15.4360

0.5008

0.00

14.9480

0.4851

1.92

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.4950

0.1644

67.20

66.34

0.83

1.25

4.6220

0.1690

66.27

4.7210

0.1726

65.55

Test Item

13.9810

0.5102

0.00

0.76

1.20

158.59

13.8660

0.5060

0.13

13.5870

0.4959

2.14

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

DPRA Prediction²

0.00% PPD 6.38%

 No or Minimal Reactivity

Negative

6.38% < PPD 22.62%

Low Reactivity

Positive

22.62% < PPD 42.47%

Moderate Reactivity

42.47% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

ReactivityClass

DPRA Predictio

0.00% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Categorization of the Test Item

Prediction Model

Prediction Model 1
(Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Prediction Model 2
(Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

1.15

Minimal Reactivity

negative

1.55

Minimal Reactivity

negative

Positive Control

67.86

High Reactivity

positive

69.38

Moderate Reactivity

positive

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
DPRA
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

In the present study the test item was dissolved in isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 395.66 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cisopropanol).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (1.15%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.