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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018/09/24 - 2018/10/26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydroxyacetone
EC Number:
204-124-8
EC Name:
Hydroxyacetone
Cas Number:
116-09-6
Molecular formula:
C3H6O2
IUPAC Name:
1-hydroxypropan-2-one
Test material form:
liquid

In chemico test system

Details on the study design:
In a non-GLP pre-test, the solubility of the test item was tested. The test item was found to be soluble in acetonitrile at a target concentration of 100 mM. Therefore, acetonitrile was used as vehicle. Based on the non-GLP pre-test, a 100 mM test item solution was prepared by dissolving 22.8 mg and 22.7 mg test item in 3 mL of the solvent acetonitrile for the Cys-peptide and Lys-peptide, respectively.

Test item samples
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were pre-pared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions. A final volume of 1 mL per sample was prepared for each sample.

Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide. All three replicates for the Cys-peptide were turbid after incubation. They were centrifuged (10 min, 400 g) and only the clear supernatant was used for the measurement. None of the replicates for the Lys-peptide were turbid after incubation.

Results and discussion

Positive control results:
Calculated peptide depletion values for the Cys-Peptide:
1. 75.44 %
2. 75.77 %
3. 77.21 %
mean: 76.14 %

Calculated peptide depletion values for the Lys-Peptide
1. 38.14 %
2. 37.76 %
3. 44.86 %
mean: 40.25 %

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
mean
Parameter:
other: depletion of both peptides after incubation with the test item
Value:
18.36
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Experiment 3, Replicate 1
Parameter:
other: Calculated peptide depletion values for the Cys-Peptide [%]
Value:
7.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Experiment 3, Replicate 2
Parameter:
other: Calculated peptide depletion values for the Cys-Peptide [%]
Value:
7.18
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Experiment 3, Replicate 3
Parameter:
other: Calculated peptide depletion values for the Cys-Peptide [%]
Value:
8.59
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Experiment 2, Replicate 1
Parameter:
other: Calculated peptide depletion values for the Lys-Peptide [%]
Value:
24.35
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Experiment 2, Replicate 2
Parameter:
other: Calculated peptide depletion values for the Lys-Peptide [%]
Value:
31.19
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
other: Experiment 2, Replicate 3
Parameter:
other: Calculated peptide depletion values for the Lys-Peptide [%]
Value:
31.77
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
mean
Parameter:
other: Calculated peptide depletion values for the Cys-Peptide [%]
Value:
7.62
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Run / experiment:
mean
Parameter:
other: Calculated peptide depletion values for the Lys-Peptide [%]
Value:
29.1
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
The ten proficiency chemicals listed in the guideline were tested using the analysis method. All ten proficiency chemicals showed the expected DPRA prediction and eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline (LAUS in-house study).

ACCEPTANCE OF RESULTS:
Solvent control
a) The mean peptide concentration of all solvent controls (Reference A and Reference C) were in the acceptable range of 0.50 ± 0.05 mM for both peptides.
b) The variation coefficients (RSD) of the measured values of Reference controls B and C in acetonitrile were in the acceptable range with 0.6 % for the Cys-peptide and 0.5 % for the Lys-Peptide, respectively.

positive control and test item
a) The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and < 14.9 %, respectively for the Cys-peptide.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and < 11.6 %, respectively for the Lys-peptide.
c) The standard deviation for the test item replicates was < 14.9 % for the percent cysteine depletion for the test item. The standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item.

Any other information on results incl. tables

Evaluation of results according to the Cysteine 1:10/Lysine 1:50 prediction model

 Mean peptide depletion [%]  Reactivity Class  DPRA Prediction
 0 – ≤ 6.38  Minimal  Negative
 > 6.38 – ≤ 22.62  Low  Positive
 > 22.62 – ≤ 42.47  Moderate  Positive
 > 42.47 - ≤ 100  High  Positive

The mean peptide depletion in the Cys-peptide and Lys-peptide assay was 18.36 %, therefore the test item was classified with:

DPRA Prediction: Positive

Reactivity class: Low

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Experiment 1 was invalid for both peptides. In Experiment 2 only for the Lys-peptide the acceptance criteria were fulfilled. In Experiment 3 all acceptance criteria were fulfilled; therefore, the test was considered valid. The DPRA prediction for the test item Hydroxyacetone was positive with reactivity class low according to the Cysteine 1:10/Lysine 1:50 prediction model. No observations arousing doubts concerning the accuracy of the results and the validity of the study were made

The mean peptide depletion in the Cys-peptide and Lys-peptide assay was 18.36 %, therefore the test item was classified with:
DPRA Prediction: Positive
Reactivity class: Low
Executive summary:

A GLP-compliant study according to OECD TG 442C (DPRA) was conducted. The study was performed in order to evaluate the reactivity of the test item Hydroxyacetone towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile was incubated 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide at 25 °C, and the peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Three experiments were performed. Experiment 1 was not valid, because the reference control A of the Cys-peptide was not in the range and the relative standard deviation (RSD) of the controls B and C was too high. For the Lys-peptide the peptide-depletion of the positive control was out of range.

In Experiment 2 the reference control A and the peptide depletion of the positive control for the Cys-peptide was not in the range and the relative standard deviation (RSD) of the controls B and C was too high. For the Lys-peptide experiment 2 was valid and the results are reported here.

The third experiment was valid for the Cys-peptide and the results are reported here.The invalid experiments are not reported in this report, but the raw data are kept in the GLP- archive of the test facility.

The mean peptide depletion in the Cys-peptide and Lys-peptide assay was 18.36 %, therefore the test item was classified with:

DPRA Prediction: Positive

Reactivity class: Low

In conclusion, the DPRA prediction is “positive” with low reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item hydroxyacetone possesses a low skin sensitisation potential.