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Skin sensitisation

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Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 March 2020 - 08 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The validated in chemico skin sensitization test is the DPRA assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in chemico test to be performed as part of weight of evidence.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
18 June 2019
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

1
Chemical structure
Reference substance name:
Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
EC Number:
909-017-6
Molecular formula:
C10H20O2 C9H28O
IUPAC Name:
Reaction mass of 1-hydroxydecan-3-one and 3-(hydroxymethyl)nonan-2-one and nonan-2-one
Test material form:
liquid

In chemico test system

Details on the study design:
Preparation of the Test Item
- The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.04 was used to correct for the purity of the test item.

Controls
- Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
- Positive Control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
- Co-elution Control: Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
- Reference Control: Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run. Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run. Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run. Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

Test System
- HPLC System: HPLC/DAD: Agilent Infinity 1260 II with Chromeleon 7.2 SR5; Detection: 220 nm signal for quantitation, 258 nm signal used as indicator for co-elution; Analytical Column: Zorbax SB-C18, 100 mm x 2.1 mm, 3.5 μm, Agilent Art. Nr. 861753-902; Pre-Column: Phenomenex, AJO-4286, 4.0 x 2.0 mm; Column Temperature: 30°C; Sample Temperature: 20-25°C; Run Time: 20 minutes; Gradient: Flow: 0.35 mL/min; Time%A/%B: 0 min/90/10; 10 min/75/25; 11 min/10/90; 13 min10/90; 13.5 min/90/10; Injection Volume: 4 μL
- HPLC Mobile Phase: HPLC Mobile Phase A: 0.1% ( v/v) trifluoroacetic acid in water; HPLC Mobile Phase B: 0.085% ( v/v) trifluoroacetic acid in acetonitrile
- Peptides: 18.18 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (35.25 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM; 20.13 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (37.78 mL) to reach a concentration of 0.667 mM; All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Pre-Experiments
- Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following solvents suitable for the test: acetonitrile, dist. water, dist. water : acetonitrile 1:1 (v/v), isopropanol. The test item was not soluble in acetonitrile, dist. water and dist. water : acetonitrile 1:1 (v/v). The test item was completely soluble in isopropanol, therefore, isopropanol was chosen as suitable vehicle for the main experiments.

Experimental Procedure
- Incubation of the Test Item with the Cysteine and Lysine Peptide: 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 following HPLC procedure.
- Preparation of the HPLC Standard Calibration Curve: A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile: 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed, resulting in 7 calibration solutions (0.534, 0.267, 0.134, 0.067, 0.033, 0.017, 0.000 mM).
- Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days, all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Data analysis
- The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions. The percent peptide depletion (PPD) was calculated according to the following formula: PPD = (1-(Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Reference Control C))*100. The absorbance at λ = 258 nm was also monitored for the samples of the test item and the reference controls as a co-elution control. The ratio of the peak areas (220 nm / 258 nm) was checked for consistency between reference control and test item samples. If this ratio was not consistent, a co-elution was assumed and the evaluation would be adjusted accordingly. Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser, if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as “0” when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration different from 100 mM. By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model (See Any other information on materials and methods) for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin, if the mean depletion of both peptides is below 6.38%.
- In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible. If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive". In cases where the co-elution occurs only with the lysine peptide, prediction model 2 can be applied (cysteine 1:10 prediction model, See Any other information on materials and methods). A single HPLC analysis for both the cysteine and the lysine peptide should be sufficient for a test chemical when the result is unequivocal. However, in cases of results close to the threshold used to discriminate between positive and negative results (i.e. borderline results), additional testing may be necessary. In situations where the mean percent depletion falls in the range of 3% to 10% for the cysteine, 1:10/lysine, 1:50 prediction model or the cysteine percent depletion falls in the range of 9% to 17% for the cysteine 1:10 prediction model, a second run should be considered, as well as a third one in case of discordant results between the first two runs.

Acceptance Criteria
- The run meets the acceptance criteria if: the standard calibration curve has a r² > 0.99, the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%, the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%, the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM, the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
- The results of the test item meet the acceptance criteria if: the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD), the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD), the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
run/experiment 1
Parameter:
mean cystein depletion
Value:
36.01 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Run / experiment:
run/experiment 1
Parameter:
mean lysine depletion
Value:
2.98 %
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
Pre-Experiments
- Solubility of the test item was determined prior to the main experiment. The test item was soluble in isopropanol. No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using isopropanol. All test item solutions were freshly prepared immediately prior to use.

Precipitation and Phase Separation
- All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item no turbidity or precipitation 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 no turbidity or precipitation 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 any of the samples.

Co-elution with the Peptide Peaks
- No relevant co-elution of the test item with any of the peptide peaks was observed. To detect a co-elution of the test item with the peptide peak, a ratio of the 220 nm peak area and the 258 nm peak area was calculated too (peak purity indication). If the ratio of the control samples and the test item samples do not differ more than 10% from each other, no sign for a co-elution is given. For the cysteine measurement the peak purity indication was < 10% (5.8 % test item replicate 1; 6.0 % test item replicate 2; 1.3 % test item replicate 3; mean: 4.37 %) and for the lysine measurement the peak purity indication was < 10% (0.3 % test item replicate 1; 2.0 % test item replicate 2; 0.1 % test item replicate 3; mean: 0.8 %).

See Tables in "any other information on results incl. tables" for acceptability criteria.

Any other information on results incl. tables

Table 3: Depletion of the Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration

Peptide Depletion

Mean Peptide Depletion

SD of Peptide Depletion

CV of depletion

mAU

mM

%

%

%

%

Positive control

4.8870

0.1599

68.73

69.27

0.92

1.33

4.8860

0.1598

68.74

4.6380

0.1518

70.33

Test item

9.9700

0.3255

36.04

36.01

0.65

1.80

10.0780

0.3291

35.35

9.8760

0.3225

36.64

 

Table 4: Depletion of the Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration

Peptide Depletion

Mean Peptide Depletion

SD of Peptide Depletion

CV of depletion

mAU

mM

%

%

%

%

Positive control

3.7970

0.1668

66.62

66.30

0.65

0.97

3.7870

0.1663

66.71

3.9190

0.1721

65.55

Test item

11.0580

0.4853

3.82

2.98

0.78

26.21

11.1690

0.4902

2.86

11.2360

0.4932

2.27

 

Table 5: Acceptance Criteria for Cysteine Peptide

Acceptance Criterion

Range

Value

pass/fail

coefficient of determination

R² >0.99

1.000

Pass

mean peptide concentration of RC A

0.45 ≤ x ≤ 0.55 mM

0.5229

Pass

mean peptide concentration of RC C (PC)

0.45 ≤ x ≤ 0.55 mM

0.5100

Pass

mean peptide concentration of RC C (TI)

0.45 ≤ x ≤ 0.55 mM

0.5086

Pass

CV of the peak area of RC B

<15%

0.58

Pass

CV of the peak area of RC C (PC)

<15%

0.15

Pass

CV of the peak area of RC C (TI)

<15%

0.77

Pass

mean peptide depletion of the PC

60.8% < x < 100%

69.27

Pass

SD of peptide depletion of the PC replicates

<14.9%

0.92

Pass

SD of peptide depletion of the TI replicates

<14.9%

0.65

Pass

 

Table 6: Acceptance Criteria for Cysteine Peptide

Acceptance Criterion

Range

Value

pass/fail

coefficient of determination

R² >0.99

1.000

Pass

mean peptide concentration of RC A

0.45 ≤ x ≤ 0.55 mM

0.4987

Pass

mean peptide concentration of RC C (PC)

0.45 ≤ x ≤ 0.55 mM

0.4993

Pass

mean peptide concentration of RC C (TI)

0.45 ≤ x ≤ 0.55 mM

0.5046

Pass

CV of the peak area of RC B

<15%

0.82

Pass

CV of the peak area of RC C (PC)

<15%

0.36

Pass

CV of the peak area of RC C (TI)

<15%

1.93

Pass

mean peptide depletion of the PC

40.2% < x < 69.0%

66.30

Pass

SD of peptide depletion of the PC replicates

<11.6%

0.65

Pass

SD of peptide depletion of the TI replicates

<11.6%

0.78

Pass

 

Applicant's summary and conclusion

Interpretation of results:
other: DPRA was positive
Conclusions:
The substance was positive in the DPRA and was classified in the “low reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.
Executive summary:

In a GLP-compliant OECD guideline 442C study, the Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. The test substance was dissolved in isopropanol, based on the results of the pre-experiments. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution and the lysine peptide solution, respectively. 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. 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. The 100 mM stock solution of the test item showed low reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (19.50%). Based on the prediction model 1, the substance was positive in the DPRA and was classified in the “low reactivity class”.