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REACH

Hydroxyacetone

EC number: 204-124-8 | CAS number: 116-09-6

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

gene mutation in bacterial cells

Ames test I (OECD 471, GLP)

plate incorporation method (Andres 2018 1a)

S. typhimurium TA97a, TA98, TA 100, TA102 and TA1535, with and without metabolic activation: negative

pre-incubation method (Andres 2018 1b)

S. typhimurium TA97a, TA98, TA102 and TA1535, with and without metabolic activation: negative

S. typhimurium TA100, with and without metabolic activation: positive

Ames test II (OECD 471, GLP)

plate incorporation method (Andres 2018 2a)

S. typhimurium TA97a, TA98, TA100 and TA1535, E. coli (WP2) with and without metabolic activation: negative

pre-incubation method (Andres 2018 2b)

S. typhimurium TA98, TA100 and TA1535, E. coli (WP2) with and without metabolic activation: negative

S. typhimurium TA97a, with and without metabolic activation: positive

cytogenicity in mammalian cells (Béres 2018)

Chromosome aberration test (OECD 473, GLP), V79 chinese hamster lung fibroblast, with and without metabolic activation: positive

gene mutation in mammalian cells (Frühmesser 2018)

HPRT test (OECD 476, GLP), V79 chinese hamster lung fibroblast, with and without metabolic activation, Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on X-chromosome: negative

Link to relevant study records

Reference 1

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: 2018/02/14-2018/02/22
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: according to guideline
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:: according to guideline
Guideline:: EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:: yes (incl. QA statement)
Remarks:: Landesamt für Umwelt, 55116 Mainz (15.05.2018)
Type of assay:: bacterial reverse mutation assay
Specific details on test material used for the study:: The test item was stored in the test facility in a closed vessel at room temperature (16.2 –23.0°C).
Target gene:: please refer to table 1
Species / strain / cell type:: S. typhimurium TA 97
Remarks:: a
Species / strain / cell type:: S. typhimurium TA 98
Species / strain / cell type:: S. typhimurium TA 100
Species / strain / cell type:: S. typhimurium TA 102
Species / strain / cell type:: S. typhimurium TA 1535
Metabolic activation:: with and without
Metabolic activation system:: S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally
Test concentrations with justification for top dose:: 5, 1.5, 0.5, 0.15, 0.05 µL/plate
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: DMSO; water
- Justification for choice of solvent/vehicle: According to the study plan, demin. water should have been used as vehicle. Accidently, in the first experiment DMSO was used as vehicle. Therefore, in the second experiment DMSO was used as vehicle, too. This can be seen as uncritical, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: sodium azide; benzo(a)pyrene; other: 4-nitro-1,2-phenylene diamine, 2-Amino-anthracene
Details on test system and experimental conditions:: METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 24 h

SELECTION AGENT (mutation assays): histidine, ampicillin, UV-radiation, crystal violet solution

NUMBER OF REPLICATIONS: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (quotient titre/toxicity)
- Any supplementary information relevant to cytotoxicity: Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.
The toxicity of the following concentration was tested: 5μL/plate. Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.
Evaluation criteria:: The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Species / strain:: S. typhimurium TA 97
Remarks:: a
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 100
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 102
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the first experiment, the test item showed no precipitates on the plates in all tested concentrations.
- Other confounding effects: No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory). All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all (one exception) were within the historical control data ranges.
Conclusions:: The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. To verify this result, a further experiment was performed.
Executive summary:: The mutagenic potential of Hydroxyacetone with Baterial Reverse Mutation Test was conducted following OECD guideline 471 and EU guideline B.13/14 and in compliance with GLP criteria. The test item Hydroxyacetone was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) using the plate incorporation method. The test item (dissolved in DMSO) was tested up to concentrations of 5 μL/plate in the strains TA97a, TA98, TA100, TA102 and TA1535. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. As a result of this experiment none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018/04/11-2018/04/20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, 55116 Mainz (15.05.2018)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test item was stored in the test facility in a closed vessel at room temperature (20±5°C)

Target gene:

Please refer to table 1

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 97

Remarks:

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally

Test concentrations with justification for top dose:

0.05, 0.15, 0.5, 1.5, 5 µL/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: water was chosen as vehincle, as there were suspicions coming from the sponsor that the test item might react with DMSO, a solvent used in another recently performed Ames test in which a positive response in TA100 with and without S9 mix under pre-incubation conditions was noted (study 17100906G803 performed at LAUS GmbH, too)

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

methylmethanesulfonate

other: 4-Nitro-1,2-phenylene diamine, 2-Amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 24 h

SELECTION AGENT (mutation assays): histidine, tryptophan, ampicillin, UV radiation, crystal violet solution

NUMBER OF REPLICATIONS: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (quotient titre/toxicity)
The toxicity of the following concentration was tested: 5 μL/plate. Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Species / strain:

S. typhimurium TA 97

Remarks:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In all experiments, no precipitation of the test item Hydroxyacetone was observed at any of the tested concentrations up to 5 μL/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

table 2 Mean Revertants first experiment

Strain		TA97a		TA98		TA100		E.coli		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin.	Mean	82	98	41	35	106	110	136	147	16	10
water	sd	8.6	21.0	5.0	7.6	10.6	10.7	41.0	8.1	2.5	3.5
DMSO	Mean	83	95	41	36	106	99	151	145	14	16
DMSO	sd	8.4	16.8	17.0	0.6	9.7	12.3	11.0	11.4	3.0	2.1
Positive Controls*	Mean	595	1001	1001	188	609	1001	1001	1001	257	131
	sd	16.7	0.0	0.0	4.0	23.1	0.0	0.0	0.0	48.2	44.6
	f(I)	7.17	10.54	24.41	5.22	5.75	10.11	6.63	6.90	16.06	8.19
5 µL/plate	Mean	82	89	38	48	91	109	136	141	20	14
	sd	9.7	16.0	4.9	3.2	22.5	23.4	7.2	7.0	2.9	1.7
	f(I)	1.00	0.91	0.93	1.37	0.86	0.99	1.00	0.96	1.25	1.40
1.5 µL/plate	Mean	89	101	36	41	85	104	143	155	15	16
	sd	24.3	19.8	2.1	0.6	7.0	14.0	15.1	13.6	3.0	3.2
	f(I)	1.09	1.03	0.88	1.17	0.80	0.95	1.05	1.05	0.94	1.60
0.5 µL/plate	Mean	89	87	42	39	95	95	139	145	14	13
	sd	20.3	14.2	4.6	7.0	18.7	11.0	5.7	10.7	4.0	2.5
	f(I)	1.09	0.89	1.02	1.11	0.90	0.86	1.02	0.99	0.88	1.30
0.15 µL/plate	Mean	76	78	36	42	98	113	149	147	12	16
	sd	13.0	5.6	4.0	5.9	13.5	11.7	10.2	4.0	1.5	3.1
	f(I)	0.93	0.80	0.88	1.20	0.92	1.03	1.10	1.00	0.75	1.60
0.05 µL/plate	Mean	91	94	38	33	101	106	149	143	14	13
	sd	20.5	9.5	3.8	2.1	2.3	5.3	6.2	7.0	3.1	2.6
	f(I)	1.11	0.96	0.93	0.94	0.95	0.96	1.10	0.97	0.88	1.30

f(I) 0 increase factor

* different positive controls were used

1001 colonies per plate means the bacteria growth was too strong for counting

Conclusions:

No increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed in all tested bacteria strains. No concentration-related increase over the tested range was found. Therefore, the test item is stated as not mutagenic under the test conditions of this experiment.

Executive summary:

This Ames test, using water as vehicle, was performed as there were suspicions coming from the sponsor that the test item might react with DMSO, a solvent used in another recently performed Ames test in which a positive response in TA100 with and without S9 mix under pre-incubation conditions was noted. The mutagenic potential of Hydroxyacetone was tested in the Salmonella typhimurium reverse mutation assay (OECD Guideline 471, GLP). In the first experiment, the test item (dissolved in demin. water) was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, Escherichia coli and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both, the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Reference 3

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: 2018/02/14-2018/02/22
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: according to guideline
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:: according to guideline
Guideline:: EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:: yes (incl. QA statement)
Remarks:: Landesamt für Umwelt, 55116 Mainz (15.05.2018)
Type of assay:: bacterial reverse mutation assay
Specific details on test material used for the study:: The test item was stored in the test facility in a closed vessel at room temperature (16.2 –23.0°C).
Target gene:: please refer to table 1
Species / strain / cell type:: S. typhimurium TA 97
Remarks:: a
Species / strain / cell type:: S. typhimurium TA 98
Species / strain / cell type:: S. typhimurium TA 100
Species / strain / cell type:: S. typhimurium TA 102
Species / strain / cell type:: S. typhimurium TA 1535
Metabolic activation:: with and without
Metabolic activation system:: S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally
Test concentrations with justification for top dose:: 5, 2.5, 1.25, 0.63, 0.31, 0.16, 0.08 µL/plate
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: DMSO; water
- Justification for choice of solvent/vehicle: According to the study plan, demin. water should have been used as vehicle. Accidently, in the first experiment DMSO was used as vehicle. Therefore, in the second experiment DMSO was used as vehicle, too. This can be seen as uncritical, because the test item was sufficiently soluble and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: sodium azide; benzo(a)pyrene; other: 4-nitro-1,2-phenylene diamine, 2-amino-anthracene
Details on test system and experimental conditions:: METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 24 h

SELECTION AGENT (mutation assays): histidine, ampicillin, UV-radiation, crystal violet solution

NUMBER OF REPLICATIONS: 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (quotient titre/toxicity)
- Any supplementary information relevant to cytotoxicity: Performed in experiment 1 only analogously to the titre control with the maximum dose of test item on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ±1°C.
The toxicity of the following concentration was tested: 5μL/plate. Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.
Evaluation criteria:: The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Species / strain:: S. typhimurium TA 100
Metabolic activation:: with and without
Genotoxicity:: positive
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 97
Remarks:: a
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 102
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
- Other confounding effects: No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
Whereas a concentration-dependent response was noted in the presence of S9 the effects seen in the absence of S9 did not reveal a concentration dependency.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) )
All strains met the criterion of at least 10E9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Conclusions:: Based on the results of this study it is concluded that Hydroxyacetone is mutagenic in the Salmonella typhimurium strain TA100 (it can be assumed that this effect is indicative for a base-pair substitution) in the absence and presence of metabolic activation under the experimental conditions in this study. There was however some suspicion coming from the sponsor of a potential interaction of the test item with DMSO. As the substance is also well soluble in water a new test with water as solvent was performed to clarify if the effects noted could be caused by the test item or by a potential reaction product of the test item with DMSO.
Executive summary:: The mutagenic potential of Hydroxyacetone with Baterial Reverse Mutation Test was conducted following OECD guideline 471 and EU guideline B.13/14 and in compliance with GLP criteria. The test item Hydroxyacetone was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation. Based on the results of the first experiment, the test item was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in all bacteria strains using the preincubation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at none of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that the test item caused an increase in the number of revertants in the bacteria strain TA100 compared to the solvent control, in the absence and presence of metabolic activation at three concentrations (5, 2.5 and 1.25 μL/plate) exceeding the threshold value of a two fold increase under both conditions. However, whereas a concentration-dependent response was noted in the presence of S9 the effects seen in the absence of S9 did not reveal a concentration-dependency.

Reference 4

Endpoint:: in vitro gene mutation study in bacteria
Type of information:: experimental study
Adequacy of study:: weight of evidence
Study period:: 2018/04/11-2018/04/20
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Reason / purpose for cross-reference:: reference to same study
Reason / purpose for cross-reference:: reference to other study
Reason / purpose for cross-reference:: reference to other study
Qualifier:: according to guideline
Guideline:: OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:: no
Qualifier:: according to guideline
Guideline:: EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:: yes (incl. QA statement)
Remarks:: Landesamt für Umwelt, 55116 Mainz (15.05.2018)
Type of assay:: bacterial reverse mutation assay
Specific details on test material used for the study:: The test item was stored in the test facility in a closed vessel at room temperature (20±5°C)
Target gene:: please refer to table 1
Species / strain / cell type:: S. typhimurium TA 1535
Species / strain / cell type:: S. typhimurium TA 97
Remarks:: a
Species / strain / cell type:: S. typhimurium TA 98
Species / strain / cell type:: S. typhimurium TA 100
Species / strain / cell type:: E. coli WP2
Metabolic activation:: with and without
Metabolic activation system:: S9: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally
Test concentrations with justification for top dose:: 0.16, 0.31, 0.63, 1.25, 2.5, 5
Vehicle / solvent:: - Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: water was chosen as vehicle, as there were suspicions coming from the sponsor that the test item might react with DMSO, a solvent used in another recently performed Ames test in which a positive response in TA100 with and without S9 mix under pre-incubation conditions was noted (study 17100906G803 performed at LAUS GmbH, too)
Untreated negative controls:: yes
Negative solvent / vehicle controls:: yes
True negative controls:: no
Positive controls:: yes
Positive control substance:: sodium azide; benzo(a)pyrene; methylmethanesulfonate; other: 4-nitro-1,2-phenylene diamine, 2-amino-anthracene
Details on test system and experimental conditions:: METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 24 h

SELECTION AGENT (mutation assays): histidine, tryptophan, ampicillin, UV radiation, crystal violet solution

NUMBER OF REPLICATIONS: 3 replicates
Evaluation criteria:: The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
: Key result
Species / strain:: S. typhimurium TA 97
Remarks:: a
Metabolic activation:: with and without
Genotoxicity:: positive
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 1535
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 98
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: S. typhimurium TA 100
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Species / strain:: E. coli WP2
Metabolic activation:: with and without
Genotoxicity:: negative
Cytotoxicity / choice of top concentrations:: cytotoxicity
Vehicle controls validity:: valid
Untreated negative controls validity:: valid
Positive controls validity:: valid
Additional information on results:: TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the second experiment, the test item showed no precipitates on the plates in all tested concentrations.
- Other confounding effects: No signs of toxicity towards the bacteria strains could be observed. The bacterial background lawn was visible and not affected. The number of revertant colonies was not reduced.
An increase in the number of revertants in the treatments with and without metabolic activation was noted under this test condition for the bacteria strains TA98, TA100 and TA1535. The increase factor was clearly below the threshold of 2.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
Conclusions:: Based on the results of this study it is concluded that Hydroxyacetone is mutagenic in the Salmonella typhimurium strain TA97a in the absence and presence of metabolic activation, but it is not mutagenic in the Salmonella typhimurium strains TA98, TA100 and TA1535 and in the Escherichia coli (WP2) strain in the absence and presence of metabolic activation under the experimental conditions used in this study. Overall, taken the findings of both studies together, there are indications for a mutagenic potential of Hydroxyacetone in the Ames test.
Executive summary:: This Ames test, using water as vehicle, was performed as there were suspicions coming from the sponsor that the test item might react with DMSO, a solvent used in another recently performed Ames test in which a positive response in TA100 with and without S9 mix under pre-incubation conditions was noted (study 1710096G803 performed at LAUS GmbH, too). The mutagenic potential of Hydroxyacetone was tested in the Salmonella typhimurin reverse mutation assay (OECD Guideline 471, GLP) with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and -S9 standing for absence of metabolic activation using demin. water as solvent. Based on the first experiment, the test item was tested up to concentrations of 5 μL/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that the test item caused an increase in the number of revertants in the bacteria strain TA97a (it can be assumed that this effect is indicative for a frame-shift mutation event) compared to the solvent control, in both the absence and presence of metabolic activation. An increase in the number of revertants in the treatments with and without metabolic activation was noted under this test condition for the bacteria strains TA98, TA100 and TA1535. But the increase factor was clearly below the threshold of 2. However, the effect noted in TA100 (it can be assumed that this effect is indicative for a base-pair substitution) in the study 17100906G803 (performed also at LAUS GmbH with the same test item) using DMSO as solvent was not reproduced.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018/05/30-2018/07/06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI - The National Institute of Pharmacy and Nutrition (21.04.2016)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

n/a

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures)
- Suitability of cells: well-established, with low background abberations
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Methods for maintenance in cell culture if applicable: The cell stocks were kept in liquid nitrogen and were routinely checked for mycoplasma infections. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2
- Modal number of chromosomes: diploid, 2n = 22
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2. DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL test item
with S9 mix: 250, 500, 1000 and 2000 μg/mL test item
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 μg/mL test item
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 125, 250, 500 and 1000 μg/mL test item
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 250, 500, 1000 and 2000 μg/mL test item
Clear cytotoxicity of about 50% was observed at the highest concentration of 1000 μg/mL after treatment with the test item for 20 hours in the absence of metabolic activation. After 3-hours treatment with test item in the absence and in the presence of metabolic activation lower than 50% of cytotoxicity was recorded at 2000 μg/mL (the maximum recommended concentration, OECD Guideline 473 (2016).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DME (Dulbecco’s Modified Eagle’s) medium as solvent
- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 92 x 17 mm dishes (for tissue cultures in TC sterile quality) at 5 x 10E5 cells each and were incubated for 24 hours in 10 mL of DME (Dulbecco’s Modified Eagle’s) medium containing 10% foetal bovine serum.
DURATION
- Exposure duration: 3h (±S9) or 20h (-S9)
- Expression time (cells in growth medium): Sampling was made at 20 hours after start of 3 h treatment (approximately 1.5 normal cell cycles from the beginning of treatment) or sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.
- Fixation time (start of exposure up to fixation or harvest of cells):

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.2 µg/mL)

STAIN (for cytogenetic assays): 5% Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were used at each test item concentration and the negative control cultures as well as the positive controls for treatment without and with S9 mix.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5% Giemsa for subsequent scoring of chromosome aberration frequencies.
All slides were independently coded before microscopic analysis and scored blind. 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

NUMBER OF CELLS EVALUATED: 300 well-spread metaphase cells (150 metaphases/slide) per test item concentration

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well-spread metaphase cells (150 metaphases/slide) per test item concentration

DETERMINATION OF CYTOTOXICITY
- Method: based on cell counts the Relative Increase in Cell Counts (RICC) was calculated

Rationale for test conditions:

according to guideline

Evaluation criteria:

Evaluation of Results:

Treatment of results
– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.
Both biological and statistical significance are considered together.
There is no requirement for verification of a clearly positive or negative response.

Statistics:

For statistical analysis the CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control were calculated with C-chart.
The data were checked for a linear trend in number of cells with aberrations (without gaps) with treatment dose using the adequate regression analysis by Microsoft Excel software.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

dose-dependent 500, 1000 and 2000 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

solvent control

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

solvent control

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

solvent control

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Remarks:

solvent control

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no relevant changes in pH after treatment with the test item
- Effects of osmolality: no relevant changes osmolality after treatment with the test item
- Precipitation: No precipitation of the test item was observed at any of the applied concentrations.

RANGE-FINDING/SCREENING STUDIES:
Solubility and Dose Selection
A clear solution of Hydroxyacetone was obtained DME (Dulbecco’s Modified Eagle’s) medium up to a concentration of 100 mg/mL. There was no precipitation in the medium at any concentration tested.
A pre-test on cytotoxicity was performed as part of this study to establish an appropriate concentration range for the main chromosome aberration assays (experiment A and B), both in the absence and in the presence of a metabolic activation (rodent S9 mix).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and Cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations 33-36 and 38 /150 cells as compared to solvent controls and were compatible with the historical positive control data of 26-44 /150 cells and 24-46 /150 cells respectively.
- Negative (solvent/vehicle) historical control data: The number of aberrations found in the solvent controls (3-4/150 cells) was in the range of historical laboratory control data (2-5/150 cells).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: RICC
- Other observations when applicable: Clear cytotoxicity of about 50% was observed at the highest concentration of 1000 μg/mL after treatment with the test item for 20 hours in the absence of metabolic activation. After 3-hours treatment with test item in the absence and in the presence of metabolic activation lower than 50% of cytotoxicity was recorded at 2000 μg/mL (the maximum recommended concentration)

Table1: RESULTS OF V79/CHROMOSOME ABERRATION ASSAY - EXPERIMENT A (3-hour treatment without S9 mix / 20-hour sampling time)

Study code: 919-473-3793 Test item : Hydroxyacetone

Date of treatment: '2018.05.30 Batch No. : Q141301 without S9 Treatment period : 3 hours

Solvent: DME medium Harvesting time : 20 h starting from the treatment

NONACTIVATION TEST CONDITION	RICC	Cytotoxicity	SCORED META-PHASES	NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		ABERRATIONS
				NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		CHROMOSOME			CHROMATID			OTHERS c

				gap+	gap-	gap-	gap+	gap-	gap	del	exchange	gap	del	exchange
Solvent Control a	100	0	150	6	4	2.667	6	4	2	0	2	0	1	1	-
Solvent Control b			150	9	4	2.667	9	4	3	1	1	2	1	1	-
Mean				8	4	2.667	8	4
Pos. Control a	47	53	150	36	29	19.333	53	40	9	7	9	4	11	13	-
Pos. Control b			150	37	31	20.667	51	34	5	4	7	12	13	10	-
Mean				37**	30**	20.000	52**	37**
TEST ITEM
250 µg/mL a	95	5	150	11	4	2.667	11	4	4	0	0	3	4	0	-
250 µg/mL b			150	10	3	2.000	11	3	6	0	1	2	1	1	-
Mean				11	4	2.333	11	4
500 µg/mL a	78	22	150	16	6	4.000	19	8	2	2	0	9	6	0	-
500 µg/mL b			150	12	6	4.000	12	6	2	1	1	4	4	0	-
Mean				14	6	4.000	16	7
1000 µg/mL a	73	27	150	14	6	4.000	14	6	3	1	0	5	2	3	-
1000 µg/mL b			150	17	9	6.000	20	11	3	2	0	6	3	6	-
Mean				16	8	5.000	17*	9
2000 µg/mL a	62	38	150	29	21	14.000	49	36	0	2	2	13	14	18	2c
2000 µg/mL b			150	24	17	11.333	36	26	4	0	2	6	9	15	1c
Mean				_27**	_19**	12.667	43**	31**

Solvent control : DME medium

Positive control : Ethyl methanesulfonate (1.0 µL/mL)

gap+ : including gaps

gap- : excluding gaps

RICC = Relative Increase in Cell Counts

del : deletion

Parallels for aberr. : a, b

** : = p < 0.01 to the concurrent negative control and to the historical control

* : = p < 0.05

c: endoreduplicated metaphase

Table 2: RESULTS OF V79/CHROMOSOME ABERRATION ASSAY - EXPERIMENT A (3-hour treatment with S9 mix / 20-hour sampling time)

Study code:919-473-3793 Test item : Hydroxyacetone

Date of treatment: '2018.05.30 Batch No. : Q141301 with S9 Treatment period : 3 hours

Solvent: DME medium Harvesting time : 20 h starting from the treatment

ACTIVATION TEST CONDITION	RICC	Cytotoxicity	SCORED META-PHASES	NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		ABERRATIONS
				NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		CHROMOSOME			CHROMATID			OTHERS c
				gap+	gap-	gap-	gap+	gap-	gap	del	exchange	gap	del	exchange
Solvent Control a	100	0	150	7	4	2.667	7	4	3	0	1	0	1	2	-
Solvent Control b			150	8	4	2.667	8	4	0	0	1	4	3	0	-
Mean				8	4	2.667	8	4
Pos.Control a	48	52	150	47	44	29.333	83	61	10	6	6	12	22	27	-
Pos.Control b			150	48	36	24.000	76	55	7	5	8	14	8	34	-
Mean				48**	_40**	26.667	80**	58**
TEST ITEM
250 µg/mL a	95	5	150	8	4	2.667	8	4	3	0	0	1	2	2	-
250 µg/mL b			150	7	4	2.667	7	4	2	0	3	1	1	0	-
Mean				8	4	2.667	8	4
500 µg/mL a	74	26	150	8	4	2.667	10	6	1	0	0	3	4	2	-
500 µg/mL b			150	9	4	2.667	9	4	1	0	1	4	2	1	-
Mean				9	4	2.667	10	5
1000 µg/mL a	69	31	150	8	4	2.667	9	4	2	0	0	3	3	1	-
1000 µg/mL b			150	12	5	3.333	13	6	4	1	0	3	0	5	-
Mean				10	5	3.000	11	5
2000 µg/mL a	63	37	150	8	3	2.000	9	4	2	0	0	3	2	2	1c
2000 µg/mL b			150	7	3	2.000	8	3	2	1	1	3	1	0	-
Mean				8	3	2.000	9	4

Solvent control : DME medium

Positive control : Cyclophosphanide (5.0 µg/mL)+ 50 µL/mL S9 mix

gap+ : including gaps

gap- : excluding gaps

RICC = Relative Increase in Cell Counts

del : deletion

Parallels for aberr. : a, b

** : = p < 0.01 to the concurrent negative control and to the historical control

c: endoreduplicated metaphase

Table 3: RESULTS OF V79/CHROMOSOME ABERRATION ASSAY - EXPERIMENT B (20-hour treatment without S9 mix / 20-hour sampling time)

Study code:919-473-3793 Test item : Hydroxyacetone

Date of treatment: '2018.05.30 Batch No. : Q141301 without S9 Treatment period : 20 hours

Solvent: DME medium Harvesting time : 20 h starting from the treatment

ACTIVATION TEST CONDITION	RICC	Cytotoxicity	SCORED META-PHASES	NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		ABERRATIONS
				NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		CHROMOSOME			CHROMATID			OTHERS c
				gap+	gap-	gap-	gap+	gap-	gap	del	exchange	gap	del	exchange	OTHERS c
Solvent Control a	100	0	150	7	3	2.000	7	3	1	0	2	3	1	0	-
Solvent Control b			150	8	4	2.667	9	4	3	1	0	2	2	1	-
Mean				8	4	2.333	8	4
Pos. Control a	50	50	150	48	42	28.000	71	47	8	5	5	16	13	24	-
Pos. Control b			150	48	44	29.333	82	57	8	9	9	17	14	25	-
Mean				48**	43**	28.667	_77**	52**
TEST ITEM
125 µg/mL a	96	4	150	7	4	2.667	7	4	0	0	3	3	1	0	-
125 µg/mL b			150	8	3	2.000	9	3	3	0	1	3	2	0	-
Mean				8	4	2.333	8	4
250 µg/mL a	85	15	150	11	6	4.000	11	6	3	0	1	2	4	1	-
250 µg/mL b			150	10	4	2.667	10	4	4	0	2	2	1	1	-
Mean				11	5	3.333	11	5
500 µg/mL a	66	34	150	10	4	2.667	10	4	3	0	1	3	1	2	-
500 µg/mL b			150	8	4	2.667	9	4	3	0	2	2	2	0	-
Mean				9	4	2.667	10	4
1000 µg/mL a	47	53	150	13	8	5.333	14	8	1	2	1	5	4	1	-
1000 µg/mL b			150	15	9	6.000	21	13	1	1	2	7	4	6	-
Mean				14	9	5.667	18*	_11**

Solvent control : DME medium

Positive control : Ethyl methanesulfonate (0.4 µL/mL)

gap+ : including gaps

gap- : excluding gaps

RICC = Relative Increase in Cell Counts

del : deletion

Parallels for aberr. : a, b

* : = p < 0.05 to the concurrent negative control and to the historical control

** : = p < 0.01 to the concurrent negative control and to the historical control

Table 4: RESULTS OF V79/CHROMOSOME ABERRATION ASSAY - EXPERIMENT B (20-hour treatment without S9 mix / 28-hour sampling time)

Study code:919-473-3793 Test item : Hydroxyacetone

Date of treatment: '2018.05.30 Batch No. : Q141301 without S9 Treatment period : 20 hours

Solvent: DME medium Harvesting time : 28 h starting from the treatment

SURVIVAL TO TREATMENT (Relative Solv. Control%)	RICC	Cytotoxicity	SCORED META-PHASES	NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		ABERRATIONS
				NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		CHROMOSOME			CHROMATID			OTHERS
				^gap+	^gap-	^gap-	^gap+	^gap-	gap	del	exchange	gap	del	exchange	OTHERS
Solvent Control a	100	0	150	8	3	2.000	8	3	2	0	1	3	2	0	-
Solvent Control b			150	6	3	2.000	6	3	2	0	2	1	1	0	-
Mean				7	3	2.000	7	3
Pos. Control a	48	52	150	45	36	24.000	63	45	9	3	8	9	10	24	-
Pos. Control b			150	41	35	23.333	65	44	6	6	7	15	13	18	-
Mean				43**	36**	23.667	64**	_45**
TEST ITEM
125 µg/mL a	97	3	150	8	3	2.000	9	3	1	1	1	5	1	0	-
125 µg/mL b			150	9	5	3.333	10	5	1	2	1	4	2	0	-
Mean				9	4	2.667	10	4
250 µg/mL a	84	16	150	10	4	2.667	10	4	2	0	2	4	2	0	-
250 µg/mL b			150	10	4	2.667	10	4	1	0	2	5	1	1	-
Mean				10	4	2.667	10	4
500 µg/mL a	66	34	150	9	4	2.667	9	4	1	0	1	4	3	0	-
500 µg/mL b			150	8	4	2.667	8	4	1	0	2	3	2	0	-
Mean				9	4	2.667	9	4
1000 µg/mL a	48	52	150	11	7	4.667	14	7	4	1	1	3	5	0	-
1000 µg/mL b			150	12	8	5.333	15	9	2	0	2	4	5	2	-
Mean				12	8	5.000	15	8

Solvent control : DME medium

Positive control : Ethyl methanesulfonate (0.4 µL/mL)

gap+ : including gaps

gap- : excluding gaps

RICC = Relative Increase in Cell Counts

del : deletion

Parallels for aberr. : a, b

** : = p < 0.01 to the concurrent negative control and to the historical control

Table 5: RESULTS OF V79/CHROMOSOME ABERRATION ASSAY - EXPERIMENT B (20 -hour treatment without S9 mix / 28 -hour sampling time)

Study code:919-473-3793 Test item : Hydroxyacetone

Date of treatment: '2018.05.30 Batch No. : Q141301 with S9 Treatment period : 3 hours

Solvent: DME medium Harvesting time : 28 h starting from the treatment

ACTIVATION TEST CONDITION	RICC	Cytotoxicity	SCORED META-PHASES	NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		ABERRATIONS
				NUMBER OF ABERRANT CELLS		% OF ABERRANT CELLS	NUMBER OF ABERRATIONS		CHROMOSOME			CHROMATID			OTHERS
				^gap+	^gap-	^gap-	^gap+	^gap-	gap	del	exchange	gap	del	exchange	OTHERS
Solvent Control a	100	0	150	8	4	2.667	8	4	0	0	0	4	4	0	-
Solvent Control b			150	8	4	2.667	8	4	1	1	2	3	1	0	-
Mean				8	4	2.667	8	4
Pos. Control a	50	50	150	46	42	28.000	83	61	8	9	12	14	11	29	-
Pos. Control b			150	49	43	28.667	83	58	11	4	9	14	11	34	-
Mean				48**	43**	28.333	83**	_60**
TEST ITEM
250 µg/mL a	94	6	150	10	4	2.667	11	5	2	0	2	4	1	2	-
250 µg/mL b			150	11	6	4.000	14	8	3	2	1	3	2	3	-
Mean				11	5	3.333	13	7
500 µg/mL a	79	21	150	12	5	3.333	12	5	4	1	1	3	1	2	-
500 µg/mL b			150	14	6	4.000	14	6	1	1	3	7	1	1	-
Mean				13	6	3.667	13	6
1000 µg/mL a	72	28	150	10	4	2.667	10	4	1	0	0	5	4	0	-
1000 µg/mL b			150	11	6	4.000	12	7	1	0	4	4	2	1	-
Mean				11	5	3.333	11	6
2000 µg/mL a	63	37	150	15	9	6.000	17	10	1	0	3	6	4	3	-
2000 µg/mL b			150	13	7	4.667	14	8	0	0	2	6	3	3	-
Mean				14	8	5.333	16	9

Solvent control : DME medium

Positive control : Cyclophosphanide (5.0 µg/mL)+ 50 µL/mL S9 mix

gap+ : including gaps

gap- : excluding gaps

RICC = Relative Increase in Cell Counts

del : deletion

Parallels for aberr. : a, b

** : = p < 0.01 to the concurrent negative control and to the historical control

Conclusions:

In conclusion, Hydroxyacetone induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung V79 cells, when tested up to maximum recommended concentration (3-hour treatment) in the absence and presence of metabolic activation and up to cytotoxic concentrations (20-hour treatment) in the absence of metabolic activation.
Thus, the test item is considered clastogenic in this system.

Executive summary:

The test item Hydroxyacetone, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a toxicity pre-test (without and with metabolic activation using rodent S9 mix), in accordance with the current OECD Guideline 473:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL test item

with S9 mix: 250, 500, 1000 and 2000 μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 125, 250, 500 and 1000 μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 125, 250, 500 and 1000 μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 250, 500, 1000 and 2000 μg/mL test item

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed at the highest concentration of 1000 μg/mL after treatment with the test item for 20 hours in the absence of metabolic activation. After 3-hours treatment with test item in the absence and in the presence of metabolic activation lower than 50% of cytotoxicity was recorded at 2000 μg/mL (the maximum recommended concentration, OECD Guideline 473 (2016)).

In experiment A in the absence of metabolic activation relevant increases in cells carrying structural chromosomal aberrations compared to concurrent control or in comparison with the histrorical control range were observed. These increases at concentrations of 500, 1000 and 2000 μg/mL were dose-dependent. The increase was statistically significant at the concentration of 2000 μg/mL.

In the experiment A in the presence of metabolic activation no statistical significant differences were observed when compared to the concurrent solvent as well as to the historical control groups.

In experiment B in the absence of metabolic activation at the dose of 1000 μg/mL relevant increases in cells carrying structural chromosomal aberrations (8 and 9 aberrant cells excluding gaps/150 cells) compared to concurrent control or in comparison with the histrorical control range were observed. These increases were not statistical significant when compared to the concurrent solvent as well as to the historical control groups, but the finding were considered biologically relevant.

In the experiment B in the presence of metabolic activation at the dose of 2000 μg/mL biologically relevant increase in cells carrying structural chromosomal aberrations (8 aberrant cells excluding gaps/150 cells) compared to concurrent control or in comparison with the historical control range was observed. There were no increases in the rate of polyploid metaphases in either experiment in the presence or absence of metabolic activation.

Endoreduplicated metaphases was observed with very low incidence (0.5-1.5/150 cells) in Experiment A.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Thus, the test item is considered clastogenic in this system.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018/05/08-2018/07/17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, 55116 Mainz (15.05.2018)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

The test item was stored in the test facility at room temperature (18.2 – 22.1 °C) protected from light.

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on X-chromosome

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: CLS (Eppelheim, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens. Especially the high proliferation rate and a high cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Cell cycle length, doubling time or proliferation index: doubling time 12 – 16 h in stock cultures
- Methods for maintenance in cell culture if applicable: Prior to use in the experiments, the cells were cleansed of pre-existing mutant cells by culturing in HAT medium (medium containing Hypoxanthine, Aminopterin and Thymidine). Cleansed and for mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous working stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
The cells were thawed 6-8 d prior treatment and cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2. The normal cell cycle time as well as the detection of the spontaneous mutant frequency of the used master cell stock is checked in each experiment. The data are stored in the archive of LAUS GmbH.
- Modal number of chromosomes: modal chromosome number of 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: DMEM complete culture medium with 5 % HS at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

experiment I: 20, 10, 5, 2.5, 1.25, 0.63 mg/mL
experiment II: 2, 1, 0.5, 0.25, 0.13, 0.06 mg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: (liquid test item) DMEM as solvent, (DMSO used for positive control DMBA)

Untreated negative controls:

yes

Remarks:

solvent

Negative solvent / vehicle controls:

yes

True negative controls:

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 500 ± 10 cells

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 168 h ± 15 min
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): (6-TG ) 6-Thioguanin

STAIN (for cytogenetic assays): 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution

NUMBER OF REPLICATIONS: for survival and viability duplicates; for mutagenicity 5 replicates

NUMBER OF CELLS EVALUATED: 5 * 10E5 ± 10E4 cells for mutagenicity, 500 ± 10 cells for viability

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: refer to table 1

Rationale for test conditions:

according to guideline

Evaluation criteria:

Acceptability
The gene mutation assay is considered acceptable if it meets the following criteria:
- the mutant frequency found in the solvent controls falls within the laboratory historical 95% control limit.
- the positive control substances must produce a significant increase (p < 0.05) in mutant frequency and lies in the range of the laboratory historical 95% control limit.
- two experimental conditions (+S9 and -S9) are tested unless one resulted in clear positive results.
- adequate number of cells (spontaneous MF is 5 - 20 * 10E-6 cells) and concentrations (minimum of 4) are analysable.

Classification
Providing that the study is acceptable, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is concentration-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that the study is acceptable, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical significance at the 1 % (p < 0.01) resp. 5 % level (p < 0.05) was evaluated by means of chi-square-test. The positive controls were tested at one concentration only and showed considerable increases in mutants.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None of the tested positive controls or test item concentrations provoked a critical change of the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.
- Effects of osmolality: None of the tested positive controls or test item concentrations provoked a critical change of the osmolality in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.
- Precipitation: No cytotoxicity or precipitation was observed in the treatments with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
According to the results of the pre-test in which no toxicity was noted up to the highest concentration, 6 concentrations were chosen for the main experiments and tested with and without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) )
All values remained well within the historical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Cloning efficiency (CE)
No cytotoxicity or precipitation was observed in the treatments with and without metabolic activation.
In conclusion, it can be stated that the test item Hydroxyacetone induced no cytotoxic effect in both approaches.

table 2: Summary of Results of Experiment I

	concentration	S9 mix	Treatment time	Culture	RelativeSurvival	mutant frequency per 10⁶cells	mutant frequency per 10⁶cells
	[mg/mL]		[h]				Mean
Solvent Control for Test Item		+	4	A	-	11	10
				B	-	9
Solvent Control for DMBA		+	4	A	-	7	9
				B	-	10
Positive Control (DMBA)	1.5 µg/mL	+	4	A	61.8%	140**	141**
				B	133.9%	141**
Test item	2.00	+	4	A	116.0%	18	20
				B	86.5%	21*
Test item	1.00	+	4	A	108.2%	12	19
				B	79.3%	27**
Test item	0.50	+	4	A	138.8%	16	16
				B	114.1%	17
Test item	0.25	+	4	A	131.8%	13	14
				B	96.5%	15
Test item	0.13	+	4	A	123.3%	n/e	n/e
				B	94.4%	n/e
Test item	0.06	+	4	A	94.9%	n/e	n/e
				B	119.7%	n/e
Solvent Control Test Item			4	A	-	7	9
				B	-	11
Solvent Control EMS			4	A	-	10	7
				B	-	5
Positive Control (EMS)	300 µg/mL		4	A	119.9%	73**	76**
				B	107.4%	79**
Test item	2.00		4	A	92.6%	24**	23*
				B	99.1%	21
Test item	1.00		4	A	70.9%	19*	20*
				B	86.8%	21
Test item	0.50		4	A	61.3%	12	14
				B	111.0%	15
Test item	0.25		4	A	94.6%	22**	17
				B	120.5%	12
Test item	0.13		4	A	91.9%	n/e	n/e
				B	118.4%	n/e
Test item	0.06		4	A	96.2%	n/e	n/e
				B	101.7%	n/e

n/e = not evaluated because the OECD 476 guideline requires only 4 concentrations

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

Conclusions:

It can be stated that under the experimental conditions of this study Hydroxyacetone did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.
Therefore, the test item Hydroxyacetone is considered to be “non-mutagenic under the con-ditions of the HPRT assay”.

Executive summary:

This study was performed according to OECD guideline 476 and EU-method B.17, in compliance with GLP to investigate the potential of Hydroxyacetone to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test, the concentrations for the main experiments were determined. Experiment I was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 hours without metabolic activation. The highest nominal concentration (2 mg/mL) applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test. Precipitation or turbidity of the test item was not visible in all experimental parts. Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

No dose-dependent increase in mutant colony numbers was observed in experiment I. In the approach with metabolic activation a statistically significant increase in mutant frequency was detected at the concentrations 2 mg/mL and 1 mg/mL (only in replicate B). However, all values remained well within the historical control range.

In the approach without metabolic activation a statistically significant increase in mutant frequency was detected at the concentrations 2 mg/mL and 1 mg/mL (mean values), as well as at the concentrations 2 mg/mL, 1 mg/mL and 0.25 mg/mL (replicate A). However, all values remained well within the historical control range.

Since the result of experiment I was not clearly negative or positive a second experiment (experiment II) was performed.

In experiment II again, no dose-dependent increase in mutant colony numbers was observed. No statistically significant increase in mutant colony number in comparison to the solvent control was detected up to the maximal concentration of the test item. All values remained within the historical control range. Therefore, the result of experiment II is clearly negative.

In consequence, the statistically significant differences noted in experiment I are obviously variations and an effect of the observed low mutation frequency of the respective control group.

It can be stated that under the experimental conditions of this study Hydroxyacetone did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test item Hydroxyacetone is considered to be “non-mutagenic under the conditions of the HPRT assay”.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Genetic toxicity in vivo_Vertesi, 2022: OECD 489, 474 and EU Method B.12; Rat, HAN:WIST of Wistar origin; oral gavage; genotoxic

Link to relevant study records

Reference

Endpoint:: in vivo mammalian cell study: DNA damage and/or repair
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 14 JUL 2022 - 15 NOV 2022
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Qualifier:: according to guideline
Guideline:: other: Council Regulation (EU) No 2017/735, Annex Part B, B.62: In vivo Mammalian Alkaline Comet Assay
Version / remarks:: 14 February 2017
Qualifier:: according to guideline
Guideline:: EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:: 14 February 2017
Qualifier:: according to guideline
Guideline:: OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:: 29th July, 2016
Qualifier:: according to guideline
Guideline:: OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:: 29th July, 2016
Principles of method if other than guideline:: Bowen D.E., Whitwell J.H., Lillford L., Henderson D., Kidd D., McGarry S., Pearce G., Beevers C. and Kirkland D.J.: Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test, Mutation Research 722: 7-19, 2011
GLP compliance:: yes (incl. QA statement)
Type of assay:: mammalian comet assay
Species:: rat
Strain:: Wistar
Remarks:: HAN:WIST of Wistar origin
Details on species / strain selection:: Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Justification of the species: Rats are routinely tested in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at TOXI-COOP ZRT.
Sex:: male
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Toxi-Coop ZRT. H-1103 Budapest, Cserkesz u. 90.
- Age at study initiation: 55-60 days (Young adult rats, 8-9 weeks old at the commencement of the treatment).
- Weight at study initiation: 260-276 g, the weight variation of animals involved at the start of the study did not exceed ± 20 %
- Assigned to test groups randomly: yes, under following basis: All animals were sorted according to body weight by computer and grouped according to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping was controlled by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.
- Housing: 3 animals / cage
Cage type: Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height).
Bedding: Certified laboratory wood bedding (SAFE 3/ 4-S-FASERN produced by J. Rettenmaier & Söhne GmbH+Co. KG; D-73494 Rosenberg Holzmühle 1 Germany). The cages and bedding were changed once during the acclimatization and experimental periods.
- Diet (e.g. ad libitum): received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
- Water (e.g. ad libitum): tap water from watering bottles (from municipal supply, as for human consumption, from 500 mL bottles), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): In the range of 22 ± 3 °C (the actual values: 19.0-24.4°C).
- Humidity (%): In the range of 30 – 70 % (the actual values: 33-69 %).
- Air changes (per hr): Provided by central air-condition system. The numbers of air changes per hour is higher than 10.
- Photoperiod (hrs dark / hrs light): Artificial light, 12 hours daily, from 6 a.m. to 6 p.m.
Route of administration:: oral: gavage
Vehicle:: - Vehicle(s)/solvent(s) used: distilled water (Aqua purificata)
- Concentration of test material in vehicle: 200, 100 and 50 mg/mL.
- Amount of vehicle (if gavage or dermal): 2000, 1000 and 500 mg/kg body weight/day
A constant treatment volume of 10 mL formulation/kg body weight was administered in all test item treatment groups, in the vehicle control group and in the positive control groups.
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
The planned doses were 2000, 1000 and 500 mg/kg body weight/day, consequently the test item was formulated in distilled water (Aqua purificata) in concentrations of 200, 100 and 50 mg/mL.
The formulations were prepared in the laboratory of the Test Facility just before each treatment.
Analysis of formulations (for checking of each concentration and homogeneity) was performed in the Analytical Laboratory of Test Facility according to the validated analytical method.
Duration of treatment / exposure:: 45h exposure, 48h sampled
Frequency of treatment:: The test item formulated in the vehicle, was administered in dose by oral gavage three times: once on the day 0, second on the day 1 (24 hours after first treatment) and third on the day 2 (45 hours after the first treatment).
Post exposure period:: 3-4 hours after the third treatment (doses and vehicle control) and 3-4 hours after the treatment (EMS positive control). the animals were euthanized and the cells of the target tissues were isolated for comet assay and in parallel bone marrow was sampled for micronucleus test (this meant the bone marrow was sampled at 24 h after the second dose, in the case of CP positive control at 24 h after the treatment).
Dose / conc.:: 2 000 mg/kg bw/day (nominal)
Remarks:: Test item treatment
Dose / conc.:: 1 000 mg/kg bw/day (nominal)
Remarks:: Test item treatment
Dose / conc.:: 500 mg/kg bw/day (nominal)
Remarks:: Test item treatment
Dose / conc.:: 0 mg/kg bw/day (nominal)
Remarks:: Negative control
No. of animals per sex per dose:: 5 animals per test item dose or vehicle control or positive control groups.
Control animals:: yes, concurrent vehicle
Positive control(s):: Positive control for comet assay:
Name: Ethyl methanesulfonate (EMS)
Batch Number: BCCH1105
Appearance: Colourless liquid
Expiry Date: May 27, 2023
Storage: At room temperature in tightly closed container, under inert atmosphere
Supplier/Manufacturer: SIGMA-ALDRICH
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw/d

Positive control for bone marrow micronucleus test:
Name: Cyclophosphamide monohydrate (CP)
Batch Number: MKCN3646
Appearance: White powder
Retest Date: October 31, 2023
Storage: At 2-8°C
Supplier/Manufacturer: SIGMA-ALDRICH

- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg bw/d
Tissues and cell types examined:: Target tissues for sample preparation and analysis: Stomach, liver, duodenum (comet assay); bone marrow (micronucleus test).
Details of tissue and slide preparation:: CRITERIA FOR DOSE SELECTION: For dose selection the proposals of corresponding OECD guidelines, the information provided by the Sponsor and the results of non-GLP preliminary dose range finding test (using the same species, strain, sex, and treatment regimen to be used in the main study) were taken into consideration.
In the range-finding test (based on the availably information) two animals (male rats, HAN:WIST of Wistar origin) were treated with the test item by oral administration at 10 mL/kg body weight treatment volume at the concentration of 2000 mg/kg body weight/day. The treatment was performed on three consecutive days with 24h and 21h intervals. At the chosen concentration level neither mortality nor any clinical sign or any suffering of animals were observed. Based on this information the maximum dose was 2000 mg/kg body weight/day, and in addition to the maximum dose two additional doses, 1000 and 500 mg/kg body weight/day were selected.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): In order to cover both the comet assay and micronucleus test designs, the combination protocol-design consisted of animals dosed at 0, 24 and 45 h, and sampled at 48 h (i.e., three hours after the final dose). Using this dosing regimen, only the first two doses (at 0 and 24 h) could impact upon the bone marrow micronucleus endpoint, as the final dose is given too close to the sampling time to have any effect on micronucleated polychromatic erythrocytes (MPCE) production. Bone marrow is sampled effectively at 24 h after the second dose, and is therefore in line with the conditions of OECD 474 recommendations. The bone marrow sample collection was performed 24 hours after the second treatment. As expulsion of the nucleus is known to occur within 5–10 h after the last cell division, with the loss of RNA and maturation to NCE status within a further 10–30-h period, it is currently recommended that sampling of bone marrow and subsequent analysis of the immature PCE population for MN should occur approximately 18–24 h following the final administration of test chemical. For comet assay the sampling time is a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hours after last treatment (based on the experience of the laboratory over the last years the sampling should be performed 3-4 hours after the second (in this case third) treatment. The animals were euthanized (consistent with the effective animal welfare legislation and 3Rs principles using Isofluran CP®) and the cells of the target tissues were isolated and care was taken to necropsy all animal at the same time after the last dose.

DETAILS OF SLIDE PREPARATION:
The bone marrow was obtained from one femur of the exposed rats immediately after sacrificing. The bone marrow was flushed with 5 mL of foetal bovine serum to a 15 mL centrifuge tube. After mixing, the cell suspension was concentrated by centrifugation at 2000 rpm for 10 minutes and the supernatant was discarded. Smears of the cell pellet was made on standard microscope slides. The slides were dried at room temperature overnight. Subsequently the slides were fixed in methanol for 5 minutes and allowed to air-dry. Slides were stained with 10 % Giemsa solution for 25 minutes; thereafter rinsed in distilled water. After drying at room temperature (at least 12 hours), slides were coated with EZ-Mount (TM).
The slide preparation for the Comet assay was conducted within one hour after single cell preparation. Four slides were prepared for each animal for each tissue sample, with six animals per dose group and vehicle control and for the EMS positive control group. In summary 24 slides per treatment per tissue for the test item dose groups and controls (negative, vehicle and positive).
Pre-treatment of Comet slides: According to general procedure, the conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. After gently removing, the underside of the slides were wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and allowed to dry.

METHOD OF ANALYSIS:
Analysis of Bone Marrow Slides
Prior to microscopic analysis, the prepared slide from each animal was given a code, which was covered the original animal numbers to ensure that the slides were scored without bias. Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The number of micronucleated cells were counted and frequency was expressed as percent of micronucleated cells based on the first 4000 PCEs counted in the optic field. The microscopic analysis was performed by eye.

Visualisation and Analysis of Comet Assay Slides
Every animal was euthanised and cells of the defined organs were isolated. For each animal and each tissue 4 slides were prepared (12 slides per animal, 72 slides per test item dose and negative, vehicle and EMS positive controls). Three of the four slides from five animals per test item treatments and per controls were stained and analysed (9 slides per animal, 45 slides per dose, per controls). Coded slides were stained and blind scored. The slides were examined with an appropriate magnification (200x) using fluorescent microscope (Nikon Upright Microscope Eclipse Ci-S) equipped with Nikon-INTENSILIGHT C-HGFI Precentered Fiber Illuminator an appropriate excitation filter (TRITC) and with an Andor-Zyla sCMOS camera. For image analysis, the Andor Komet GLP 7.1.0 (Andor Technology) was used. For each tissue sample, fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, per vehicle control and per EMS positive control). DNA strand breaks in the comet assay were measured by independent endpoints such as %tail DNA, olive tail moment (OTM) and tail length. The tail % DNA (also known as tail intensity) was applied for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. Additionally, the OTM and tail length values were collected. The OTM is expressed in arbitrary units and is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. Ghost cells, also known as clouds or hedgehogs, are morphological indicative of highly damaged cells and their presence is often associated with severe genotoxicity, necrosis and apoptosis. Ghost cells were excluded from the image analysis data collection, however determining of their frequency is useful for the data interpretation. The ghost cells were recorded for each slide per animal, per treatment and per tissue.
Evaluation criteria:: A study is considered as valid when:
The applied concurrent negative control is considered as acceptable for addition of the laboratory historical control databases (for comet assay and for bone marrow micronucleus test parts of this study).
Concurrent positive controls induce responses that are compatible with those generated in the historical control database and produce a statistically significant increase compared with the concurrent negative control.
Adequate numbers of cells and doses were analyzed.
The highest dose was selected according to the criteria required by the OECD 474 and 489 guidelines.
In the case of outlier assay data, they may be acceptable for inclusion in, as long as these data are not extreme outliers and there is evidence of absence of technical or human failure.
Statistics:: The statistical analysis of % tail DNA values, tail length and OTM values; the number of ghost cells; furthermore, the number of micronucleated polychromatic erythrocytes and the proportion of immature among total erythrocytes was carried out using the appropriate statistical methods, using SPSS software.
The heterogeneity of the obtained data was tested. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the inter-group comparisons were performed using Mann-Whitney U-test.
: Key result
Sex:: male
Genotoxicity:: positive
Toxicity:: yes
Vehicle controls validity:: valid
Negative controls validity:: valid
Positive controls validity:: valid
Remarks on result:: other: While the investigated test item Hydroxyacetone was not genotoxic in the examined tissues in the comet assay, it showed unequivocally genotoxic activity in the rat bone marrow micronucleus test.
Additional information on results:: RESULTS OF RANGE-FINDING STUDY
- Dose range: the maximum dose was 2000 mg/kg body weight/day, and in addition to the maximum dose two additional doses, 1000 and 500 mg/kg body weight/day were selected.
-Solubility: The test item Hydroxyacetone in distilled water formulations was considered to be homogeneous.
- Clinical signs of toxicity in test animals: At the chosen concentration level neither mortality nor any clinical sign or any suffering of animals were observed.
- Rationale for exposure: The oral route was considered to be the most relevant exposure route and for comet assay the glandular stomach, the duodenum and the liver were selected as the tissues of interest in line with the respective requirements of the EU authorities requesting this test; furthermore, bone marrow samples were obtained for analysis from one femur of the exposed rats.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): Negative control: 0.56 ± 0.07. test item 500mg/kg bw/day: 0.54 ± 0.06. test item 1000mg/kg bw/day: 0.59 ± 0.03. test item 2000mg/kg bw/day: 0.55 ± 0.05. Positive control: 0.47 ± 0.07
- Evidence of cytotoxicity in tissue analysed: Cytotoxicity was determined (as a first screening part of comet assay) on a small sample of each isolated cell
Conclusions:: The test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum and liver cells. No biologically and statistically significant increases in the frequency of MPCEs were seen in the dose groups of 500 mg/kg body weight/day compared to the negative control group, however, a biologically relevant, dose-dependent and statistically significant increase in the frequency of MPCEs were seen at dose levels of 1000 and 2000 mg/kg body weight/day compared to the concurrent vehicle control group. While the investigated test item was not genotoxic in the examined tissues in the comet assay, it showed unequivocally genotoxic activity in the rat bone marrow micronucleus test.
Executive summary:: The test item was investigated by the means of a combined assay of an in vivo comet assay on isolated stomach, duodenum and liver cells under alkaline conditions and in parallel in an in vivo bone marrow micronucleus test in the male HAN:WIST rats. The test was done according to OECD Guideline 489, 474 and EU Method B.12 under GLP compliance. The test item was administered three times via oral gavage at the dose levels 2000, 1000 and 500 mg/kg body weight/day. The applied treatment regime (dose 1 at 0-hour, dose 2 at 24 hours, dose 3 at 45 hours) as well as the samplings (e.g.: 3 hours after the last treatment for comet assay, 24 hours after the second treatment for micronucleus test) were adequate for above method combination approach. Concurrent controls confirmed the sensitivity and validity of both test parts of combined assay. Under the experimental conditions presented in this report, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum and liver cells. No biologically and statistically significant increases in the frequency of MPCEs were seen in the dose groups of 500 mg/kg body weight/day compared to the negative control group, however, a biologically relevant, dose-dependent and statistically significant increase in the frequency of MPCEs were seen at dose levels of 1000 and 2000 mg/kg body weight/day compared to the concurrent vehicle control group. While the investigated test item was not genotoxic in the examined tissues in the comet assay, it showed unequivocally genotoxic activity in the rat bone marrow micronucleus test.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed (positive)

Additional information

gene mutation in bacterial cells (Ames test)

Ames I

The mutagenic potential of Hydroxyacetone with Baterial Reverse Mutation Test was conducted following OECD guideline 471 and EU guideline B.13/14 and in compliance with GLP criteria. The test item Hydroxyacetone was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) using the plate incorporation and the pre-incubation method. The test item (dissolved in DMSO) was tested up to concentrations of 5 μL/plate in the strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of S9-mix.

plate incorporation method (Andres 2018 1a)

pre-incubation method (Andres 2018 1b)

The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at none of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that the test item caused an increase in the number of revertants in the bacteria strain TA100 compared to the solvent control, in the absence and presence of metabolic activation at three concentrations (5, 2.5 and 1.25 μL/plate) exceeding the threshold value of a two fold increase under both conditions. However, whereas a concentration-dependent response was noted in the presence of S9 the effects seen in the absence of S9 did not reveal a concentration-dependency.

Ames II

A second Ames test, using water as vehicle, was performed as there were suspicions coming from the sponsor that the test item might react with DMSO, a solvent used in the first Ames test (Ames I) in which a positive response in TA100 with and without S9 mix under pre-incubation conditions was noted. Therefore, the mutagenic potential of Hydroxyacetone was again tested in the Salmonella typhimurim reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100 and TA1535) and Escherichia coli (WP2) using the plate incorporation and pre-incubation method. The tests were performed in the presence and absence of metabolic activation up to a concentration of 5 µL/plate using demin. water as solvent.

plate incorporation method (Andres 2018 2a)

pre-incubation method (Andres 2018 2b)

The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that the test item caused an increase in the number of revertants in the bacteria strain TA97a (it can be assumed that this effect is indicative for a frame-shift mutation event) compared to the solvent control, in both the absence and presence of metabolic activation. An increase in the number of revertants in the treatments with and without metabolic activation was noted under this test condition for the bacteria strains TA98, TA100 and TA1535. But the increase factor was clearly below the threshold of 2. However, the effect noted in TA100 (it can be assumed that this effect is indicative for a base-pair substitution) in the first Ames test (Ames I) (performed also at LAUS GmbH with the same test item) using DMSO as solvent was not reproduced.

Conclusion

Overall, taken the findings of both studies together, there are indications for a mutagenic potential of the test item Hydroxyacetone in the Ames test but with regard to tester strain and mode of action the two tests give no reproducible results.

cytogenicity in mammalian cells (Chromosome aberration test) (Béres 2018)

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL test item

with S9 mix: 250, 500, 1000 and 2000 μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 125, 250, 500 and 1000 μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 125, 250, 500 and 1000 μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 250, 500, 1000 and 2000 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed at the highest concentration of 1000 μg/mL after treatment with the test item for 20 hours in the absence of metabolic activation. After 3-hours treatment with test item in the absence and in the presence of metabolic activation lower than 50% of cytotoxicity was recorded at 2000 μg/mL (the maximum recommended concentration, OECD Guideline 473 (2016).

Experiment A

In the absence of metabolic activation relevant increases in cells carrying structural chromosomal aberrations compared to concurrent control or in comparision with the historical control range were observed. These increases at concentrations of 500, 1000 and 2000 μg/mL were dose-dependent. At a concentration of 2000 μg/mL the increase was statistically significant.

In the presence of metabolic activation no statistical significant differences were observed when compared to the concurrent solvent as well as to the historical control groups.

Endoreduplicated metaphases was observed with very low incidence (0.5-1.5/150 cells).

Experiment B

In the absence of metabolic activation at the dose of 1000 μg/mL relevant increases in cells carrying structural chromosomal aberrations (8 and 9 aberrant cells excluding gaps/150 cells) compared to concurrent control or in comparision with the historical control range were observed. These increases were not statistical significant when compared to the concurrent solvent as well as to the historical control groups, but the finding were considered biologically relevant.

In the presence of metabolic activation at the dose of 2000 μg/mL biologically relevant increase in cells carrying structural chromosomal aberrations (8 aberrant cells excluding gaps/150 cells) compared to concurrent control or in comparison with the historical control range was observed.

Conclusion

There were no increases in the rate of polyploid metaphases in either experiment in the presence or absence of metabolic activation. Endoreduplicated metaphases was observed with very low incidence (0.5-1.5/150 cells) in Experiment A. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. In conclusion, Hydroxyacetone induced structural chromosome aberrations and endoreduplication in Chinese Hamster lung V79 cells, when tested up to maximum recommended concentration (3-hour treatment) in the absence and presence of metabolic activation and up to cytotoxic concentrations (20-hour treatment) in the absence of metabolic activation. Thus, the test item is considered clastogenic in this system.

Gene mutation in mammalian cells (HPRT) (Frühmesser 2018)

This study was performed according to OECD Guideline 476 and EU-Method B.17, in compliance with GLP to investigate the potential of Hydroxyacetone to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). The assay comprised a pre-test and two independent experiments (experiment I and II). The pre-test was done to detect a potential cytotoxic effect of the test item. Based on the results of this test, the concentrations for the main experiments were determined.

Experiment I was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. Experiment II was performed with a treatment period of 24 hours without metabolic activation.The highest nominal concentration (2 mg/mL) applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test. Precipitation or turbidity of the test item was not visible in all experimental parts. Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

No dose dependent increase in mutant colony numbers was observed in experiment I. In the approach with metabolic activation a statistically significant increase in mutant frequency was detected at the concentrations 2 mg/mL and 1 mg/mL (only in replicate B). However, all values remained well within the historical control range.

In the approach without metabolic activation a statistically significant increase in mutant frequency was detected at the concentrations 2 mg/mL and 1 mg/mL (mean values) as well as at the concentrations 2 mg/mL, 1 mg/mL and 0.25 mg/mL (replicate A). However, all values remained well within the historical control range.

Since the result of experiment I was not clearly negative or positive a second experiment (experiment II) was performed.

In experiment II again no dose dependent increase in mutant colony numbers was observed. No statistically significant increase in mutant colony number in comparison to the solvent control was detected up to the maximal concentration of the test item. All values remained within the historical control range. Therefore, the result of experiment II is clearly negative.

Taken all data together, no biological relevant increase in mutant colony numbers was observed in both experiments up to the maximal concentration of the test item. In consequence, the statistically significant differences noted in experiment I are obviously variations and an effect of the observed low mutation frequency of the respective control group. It can be stated that under the experimental conditions of this study Hydroxyacetone did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation. Therefore, the test item Hydroxyacetone is considered to be “non-mutagenic under the conditions of the HPRT assay”.

Genetic toxicity in vivo (Vertesi, 2022)

The test item was investigated by the means of a combined assay of an in vivo comet assay on isolated stomach, duodenum and liver cells under alkaline conditions and in parallel in an in vivo bone marrow micronucleus test in the male HAN:WIST rats. The test was done according to OECD Guideline 489, 474 and EU Method B.12 under GLP compliance. The test item was administered three times via oral gavage at the dose levels 2000, 1000 and 500 mg/kg body weight/day. The applied treatment regime (dose 1 at 0-hour, dose 2 at 24 hours, dose 3 at 45 hours) as well as the samplings (e.g.: 3 hours after the last treatment for comet assay, 24 hours after the second treatment for micronucleus test) were adequate for above method combination approach. Concurrent controls confirmed the sensitivity and validity of both test parts of combined assay. Under the experimental conditions presented in this report, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum and liver cells. No biologically and statistically significant increases in the frequency of MPCEs were seen in the dose groups of 500 mg/kg body weight/day compared to the negative control group, however, a biologically relevant, dose-dependent and statistically significant increase in the frequency of MPCEs were seen at dose levels of 1000 and 2000 mg/kg body weight/day compared to the concurrent vehicle control group. While the investigated test item was not genotoxic in the examined tissues in the comet assay, it showed unequivocally genotoxic activity in the rat bone marrow micronucleus test.

Justification for classification or non-classification

Based on the results of the combined assay of an in vivo comet assay and an in vivo bone marrow micronucleus test in the male HAN:WIST rats according to OECD Guideline 489, 474 and EU Method B.12 under GLP compliance, the registered substance is classified as Muta Cat. 2 (H341: Suspected of causing genetic defects).

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Dose (mg/kg body weight/day)	Stomach
	Measured parameters
	% Tail DNA	Tail length (µm)	OTM
Negative (vehicle) control	10.09 ± 5.84	28.79 ± 11.85	1.47 ± 1.25
500	6.15 ± 2.26	17.30 ± 9.56	0.77 ± 0.45
1000	12.86 ± 4.91	33.33 ± 10.77	1.79 ± 0.88
2000	6.25 ± 1.49	13.90 ± 5.48 * ^DN	0.65 ± 0.18
Positive (EMS) control	31.21 ± 5.62 ** ^DN	60.58 ± 12.58 ** ^DN	7.34 ± 2.61 ** ^U
Negative control ranges	0.00 – 14.96	0.00 – 35.00	0.00 – 2.63
Positive (EMS) control ranges	16.96 – 44.32	23.18 −130.47	0.00 – 23.91

Dose (mg/kg body weight/day)	Duodenum
	Measured parameters
	% Tail DNA	Tail length (µm)	OTM
Negative (vehicle) control	4.27 ± 0.70	8.26 ± 1.97	0.40 ± 0.08
500	4.37 ± 0.50	8.76 ± 1.23	0.43 ± 0.05
1000	7.30 ± 3.23	15.19 ± 10.17	0.73 ± 0.47
2000	5.72 ± 2.39	13.29 ± 9.91	0.61 ± 0.31
Positive (EMS) control	25.24 ± 3.74 ** ^U	50.73 ± 7.27 ** ^U	5.00 ± 1.17 ** ^U
Negative control ranges	1.19 – 7.13	4.23 – 11.36	0.14 – 0.63
Positive (EMS) control ranges	11.47 – 46.12	26.21 − 127.89	1.78 – 18.68

Dose (mg/kg body weight/day)	Liver
	Measured parameters
	% Tail DNA	Tail length (µm)	OTM
Negative (vehicle) control	3.08 ± 0.27	6.56 ± 0.26	0.32 ± 0.02
500	2.70 ± 0.45	6.80 ± 0.52	0.29 ± 0.04
1000	3.33 ± 0.78	7.10 ± 0.97	0.34 ± 0.08
2000	2.80 ± 0.83	7.03 ± 0.95	0.31 ± 0.07
Positive (EMS) control	27.66 ± 3.40 ** ^U	54.63 ± 10.10 ** ^U	6.30 ± 1.23 ** ^U
Negative control ranges	2.62 – 6.61	5.78 – 10.50	0.27 – 0.69
Positive (EMS) control ranges	11.06 – 34.44	34.96 −99.19	1.13 – 13.80

Dose (mg/kg body weight/day)	Mean of MPCE	Mean of PCE/PCE+NCE
Negative (vehicle) control	9.80 ± 4.49	0.56 ± 0.07
500	13.80 ± 2.59	0.54 ± 0.06
1000	17.80 ± 4.55 * ^KW	0.59 ± 0.03
2000	21.40 ± 4.04 * ^KW	0.55 ± 0.05
Positive (CP) control	81.80 ± 14.62 ** ^KW	0.47 ± 0.07 * ^DN
Negative control ranges	0.37 – 16.96	0.38 – 0.65
Positive (CP) control ranges	33.59 – 153.61	0.27 – 0.50

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin.water	Mean	69	66	32	27	78	72	225	263	21	19
Demin.water	sd	16.2	6.0	2.6	3.5	15.4	10.0	19.6	56.0	1.5	3.2
DMSO	Mean	69	95	27	28	73	78	225	287	19	21
DMSO	sd	10.4	14.0	6.0	1.2	12.0	9.5	16.7	18.9	3.8	3.6
Positive Controls*	Mean	329	657	1001	101	323	1001	692	727	257	143
	sd	99.6	79.4	0.0	24.6	54.5	0.0	36.0	47.4	14.0	23.0
	f(I)	4.77	6.92	37.07	3.61	4.14	12.83	3.08	2.53	12.24	6.81
5µL/plate	Mean	79	96	35	24	79	93	237	281	23	25
	sd	4.9	21.5	4.0	1.7	13.6	10.0	26.6	26.6	0.6	5.5
	f(I)	1.14	1.01	1.30	0.86	1.08	1.19	1.05	0.98	1.21	1.19
1.5µL/plate	Mean	66	85	21	30	85	75	268	229	22	21
	sd	5.0	12.2	3.6	8.1	10.1	8.9	26.2	30.6	1.0	4.2
	f(I)	0.96	0.89	0.78	1.07	1.16	0.96	1.19	0.80	1.16	1.00
0.5µL/plate	Mean	74	81	25	27	83	85	215	241	23	20
	sd	13.6	20.1	9.8	6.1	10.1	6.0	24.1	10.1	5.1	4.4
	f(I)	1.07	0.85	0.93	0.96	1.14	1.09	0.96	0.84	1.21	0.95
0.15 µL/plate	Mean	80	88	24	19	79	80	248	280	21	20
	sd	10.4	10.7	3.1	0.6	6.4	11.5	34.2	35.6	0.6	2.5
	f(I)	1.16	0.93	0.89	0.68	1.08	1.03	1.10	0.98	1.11	0.95
0.05 µL/plate	Mean	84	80	22	23	74	77	296	239	17	19
	sd	11.5	9.7	3.6	7.2	15.2	14.0	30.2	36.3	2.6	1.5
	f(I)	1.22	0.84	0.81	0.82	1.01	0.99	1.32	0.83	0.89	0.90

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin.water	Mean	83	81	31	35	115	116	269	244	26	22
Demin.water	Sd	14.4	8.1	5.1	4.7	7.5	8.4	12.2	4.0	4.0	3.5
DMSO	Mean	76	90	35	33	86	92	307	311	21	21
DMSO	Sd	11.6	10.6	3.5	6.9	8.4	12.5	46.9	6.1	3.5	1.5
Positive Controls*	Mean	331	313	441	104	531	1001	1320	1312	331	272
	Sd	40.1	34.0	18.9	18.0	45.5	0.0	36.7	42.3	53.3	32.0
	f(I)	4.36	3.48	12.6	3.15	4.62	10.88	4 .30	4.22	12.73	12.95
5 µL/plate	Mean	73	96	35	34	197	403	256	256	30	32
	Sd	6.6	29.0	0.6	8.5	18.6	22.0	61.6	42.1	2.5	5.2
	f(I)	0.96	1.07	1.00	1.03	2.29	4.28	0.83	0.82	1.43	1.52
2.5 µL/plate	Mean	85	116	32	40	152	272	215	216	33	29
	Sd	18.5	17.3	8.7	3.2	26.9	31.2	9.2	25.0	2.5	2.1
	f(l)	1.12	1.29	0.91	1.21	1.77	2.96	0.70	0.69	1.57	1.38
1.25 µL/plate	Mean	85	116	30	42	213	162	239	229	29	26
	Sd	16.0	15.9	7.5	5.7	41.6	77.5	37.2	28.1	5.1	3.5
	f(I)	1.12	1.29	0.86	1.27	2.48	1.76	0.78	0.74	1.38	1.24
0.63 µL/plate	Mean	87	74	31	30	145	116	227	263	30	26
	Sd	6.6	2.0	9.5	5.0	4.0	28.4	30.0	12.9	1.2	3.6
	f(I)	1.14	0.82	0.89	0.91	1.69	1.26	0.74	0.85	1.43	1.24
0.31 µL/plate	Mean	86	93	39	40	119	112	209	261	20	23
	Sd	10.2	27.5	2.6	1.2	4.2	14.4	6.1	18.9	3.8	3.0
	f(I)	1.13	1.03	1.11	1.21	1.38	1.22	0.68	0.84	0.95	1.10
0.16 µL/plate	Mean	76	75	39	42	99	100	228	252	17	21
	Sd	10.0	8.2	4.7	2.5	12.7	5.9	25.0	52.9	4.4	2.5
	f(I)	1.00	0.83	1.11	1.27	1.15	1.09	0.74	0.81	0.81	1.00
0.08 µL/plate	Mean	69	102	33	40	93	93	237	273	19	18
	Sd	10.0	15.6	5.0	4.0	17.3	16.2	26.0	68.2	1.5	3.0
	f(I)	0.91	1.13	0.94	1.21	1.08	1.01	0.77	0.88	0.90	0.86

Strain		TA97a		TA98		TA100		E.coli		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin.water	Mean	80	86	44	47	103	113	143	157	15	16
Demin.water	sd	16.6	8.4	7.2	6.9	19.0	10.1	11.4	19.7	2.0	3.8
DMSO	Mean	74	85	47	45	86	81	142	152	17	15
DMSO	sd	5.9	2.3	13.5	14.5	4.4	3.1	6.9	20.8	2.3	1.5
Positive Controls*	Mean	536	443	504	336	244	1001	1001	1001	233	76
	sd	27.7	88.1	65.5	57.7	16.0	0.0	0.0	0.0	88.9	16.0
	f(I)	7.24	5.21	10.72	7.47	2.37	12.36	7.05	6.59	15.53	5.07
5 µL/plate	Mean	239	274	84	88	166	161	118	147	27	25
	sd	62.3	62.6	21.7	6.6	8.3	6.0	37.7	9.8	4.7	6.0
	f(I)	2.99	3.19	1.91	1.87	1.61	1.42	0.83	0.94	1.80	1.56
2.5 µL/plate	Mean	288	243	59	77	155	160	139	143	22	20
	sd	36.7	35.2	21.7	12.1	5.0	4.6	18.1	41.1	1.2	2.6
	f(I)	3.6	2.83	1.34	1.64	1.50	1.42	0.97	0.91	1.47	1.25
1.25 µL/plate	Mean	88	88	51	50	105	116	140	111	17	21
	sd	10.6	17.0	15.2	19.1	12.5	12.5	5.6	21.9	0.6	2.5
	f(I)	1.10	1.02	1.16	1.06	1.02	1.03	0.98	0.71	1.13	1.31
0.63 µL/plate	Mean	75	85	46	48	105	127	124	137	21	28
	sd	7.6	4.2	15.5	14.7	16.4	21.8	23.5	8.7	3.8	7.0
	f(I)	0.94	0.99	1.05	1.02	1.02	1.12	0.87	0.87	1.40	1.75
0.31 µL/plate	Mean	87	98	45	48	119	116	122	123	26	25
	sd	16.8	27.6	3.6	0.6	6.2	8.5	7.6	14.4	10.1	4.0
	f(I)	1.09	1.14	1.02	1.02	1.16	1.03	0.85	0.78	1.73	1.56
0.16 µL/plate	Mean	98	103	41	41	111	88	122	129	22	27
	sd	21.5	10.1	5.9	13.9	23.7	15.1	19.7	39.7	9.5	8.1
	f(I)	1.23	1.20	0.93	0.87	1.08	0.78	0.85	0.82	1.47	1.69

Registration Dossier

Registration Dossier

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Diss Factsheets

Hydroxyacetone

Endpoint summary

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Link to relevant study records

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Reference 1

Reference 2

Reference 3

Reference 4

Reference 5

Reference 6

Endpoint conclusion

Genetic toxicity in vivo

Description of key information

Link to relevant study records

Reference

Endpoint conclusion

Additional information

Justification for classification or non-classification

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