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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

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
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
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.

Table 2: Mean Revertants second experiment

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

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
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

f(I) = increase factor

* different positive controls were use

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.

Reason / purpose for cross-reference:
reference to other study
Reference
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:
a
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:
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: 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:
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 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
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.

Reason / purpose for cross-reference:
reference to other study
Reference
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.

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.water   Mean 80 86 44 47 103 113 143 157 15 16  
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

 

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

f(I) 0 increase factor

* different positive controls were used

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

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.

Data source

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

Materials and methods

Test guidelineopen allclose all
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

Test material

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

Method

Target gene:
please refer to table 1
Species / strainopen allclose all
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.
Controls
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.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

Table 2: Mean Revertants First Experiment

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
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
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

f(I) = increase factor

*different positive controls were used

Applicant's summary and conclusion

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.