Hydroxyacetone

Endpoint summary

Description of key information

DPRA, OECD 442C, GLP, positive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

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)

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.

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 %

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

in vitro study

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 (mean Lys-depletion 29.1 %)

Experiment 3 was valid for the Cys-peptide (mean Cys-depletion 7.62 %).

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 pos-sesses a low skin sensitisation potential.

information from in silico methods and read-across

Profiling with the OECD QSAR Toolbox v4.2 revealed an alert associated with protein-binding (structural alert: ketones; mechanism: nucleophilic addition) identified by the profiler Protein-binding by OASIS. This is in line with the positive result of the DPRA The following data for the structural analogues acetone (CAS 67-64-1) and dihydroxyacetone (CAS 96-26-4) are available: Acetone was tested in a Guinea Pig Maximisation Test (Magnusson and Kligman) in a concentration of 100 %. No positive skin response was found in any of the treated animals (information from ECHA disseminated dossier). For dihydroxyacetone a murine Local Lymph Node Assay (OECD 429) is available. Stimulation Indices (S.l.) of 1.04, 1.16, and 0.77 were determined with the test item at concentrations of 12.5, 25, and 50% in ethanol/water (30/70 v/v), respectively. Thus, Dihydroxyacetone was not a skin sensitiser in this assay (information from ECHA disseminated dossier). QSAR profiling and the in chemico test DPRA point to a weak protein reactivity of hydroxyacetone, presumably caused by the Keto-group. On the other hand, read-across to the structural similar substances acetone and dihydroxyacetone provides substantial evidence that hydroxyacetone is not sensitising in in vivo tests. Acetone and dihydroxyacetone do also possess a keto-group, which was identified by the OECD QSAR Toolbox v4.2 as structural alert for protein-binding. Nevertheless, acetone and dihydroxyacetone were negative in two different in vivo test systems (LLNA and GPMT). This clearly indicates that the weak protein reactivity of the Keto-group does not necessarily induce a skin sensitisation response in vivo. Therefore, the positive in chemico result for hydroxyacetone is considered to be false positive. There is evidence that the in vitro/in chemico test may provide false positive results due to the weak protein-reactivity of the Keto-group. To clarify if skin sensitisation is induced in vivo, it is proposed to perform a murine local lymph node assay (LLNA), which is the first-choice method according to REACH Annex VII point 8.3.2 Column 2.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

There is evidence that the in vitro/in chemico test may provide false positive results due to the weak protein-reactivity of the Keto-group. To draw a final conclusion concerning the skin sensitisation potenial classification of hydroxyacetone, the registrant proposes to conduct a further local lymphnode assay according to OECD TG 429, which is the first-choice method according to REACH Annex VII point 8.3.2 Column 2.