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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: The reported data of the mutagenicity assay shows, that under the experimental conditions reported, the test item (acting as direct mutagen and pro-mutagen, in absence and also in the presence of exogenous metabolic activation) induced gene mutations by frameshift and base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered mutagenic in this bacterial reverse mutation assay.

HPRT: The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in the in vitro test in Chinese hamster ovary cells. Thus, the test item was not mutagenic in mammalian cells under the conditions of this study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-21 and 2015-09-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use (2012)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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:
- S9 mix: 5000; 1600; 500; 160; 50 and 16 μg/plate
+ S9 mix: 1000; 750; 500; 160; 50; 32 and 16 μg/plate
Vehicle / solvent:
- Vehicle/solvent used: acetone
- Justification for choice of solvent/vehicle: The chosen vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary solubility test.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: 4-nitro-1,2-phenylene-diamine (TA98), sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), Methyl-methanesulfonate (E.coli WP2 uvrA); +S9 mix: 2-aminoanthracene (all of Salmonella strains, E.coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration:

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: revertant colony number
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 5000 - 160 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 5000 - 500 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA98, TA1537 and E.coli WP2 uvrA
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
at 5000 and 1600 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at 750 and 500 µg/plate
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
Genotoxicity:
positive
Remarks:
at 500 and 160 µg/plate
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
Genotoxicity:
positive
Remarks:
at 160 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98
Metabolic activation:
with
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: Microdrops were observed in all test strains at 5000 and 1600 µg/plate in the absence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. The following concentrations were chosen based on the results obtained in the pre-experiment for toxicity: 5000, 1600, 500, 160, 50 and 16 µg/plate (-S9 mix); and 1000, 750, 500, 160, 50, 32 and 16 µg/plate (+S9 mix).

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: Signs of cytotoxicity (absent or reduced revertant colony numbers and/or affected background lawn development) were observed in all tested strains only in presence of metabolic activation. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The performance of an additional confirmatory experiment was considered as not necessary for the final conclusion of the study because unequivocal positive results were obtained in the Initial Mutation Test (Plate Incorporation Test).

Conclusions:
Interpretation of results: positive

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item (acting as direct mutagen and pro-mutagen, in absence and also in the presence of exogenous metabolic activation) induced gene mutations by frameshift and base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered mutagenic in this bacterial reverse mutation assay.
Executive summary:

The mutagenic potential of the test substance (50.1% purity) was tested in the Bacterial Reverse Mutation Assay in Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA. The test item was dissolved in acetone. In the Initial Mutation Test the following concentrations were examined: In absence of metabolic activation (-S9 Mix): 5000; 1600; 500; 160; 50 and 16 μg/plate; and in the presence of metabolic activation (+S9 Mix): 1000; 750; 500; 160; 50; 32 and 16 μg/plate. Due to the unequivocal demonstrative positive results obtained in the experiment I, (Plate Incorporation Test) the performance of an additional confirmatory experiment was considered as not necessary for the final conclusion of the study. The concentrations, including the controls, were tested in triplicate. In the performed experiment positive and negative (vehicle) controls were run concurrently. In the Initial Mutation Test following treatment with the test substance significant, biological relevant, dose-related changed revertant colony number increases, unequivocal positive results were noticed in the concentration range of 5000-160 μg/plate in S. typhimurium TA100, in the range of 5000-500 μg/plate in TA1535, and at 5000 and 1600 μg/plate in TA98, TA1537 and E. coli WP2 uvrA, in the absence of exogenous metabolic activation (-S9 Mix). Positive results were obtained at 750 and 500 μg/plate in E. coli WP2 uvrA, at 500 and 160 μg/plate in S. typhimurium TA100 and at 160 μg/plate in S. typhimurium TA98, with addition of metabolic activation (+S9 Mix). The mutagenic effect of the test item was unequivocal characteristic and very intensive. Signs of cytotoxicity (absent or reduced revertant colony numbers and/or affected background lawn development) were observed in all tested strains only in presence of metabolic activation. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item (acting as direct mutagen and pro-mutagen, in absence and also in the presence of exogenous metabolic activation) induced gene mutations by frameshift and base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09 till 2015-10
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)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vitro mammalian gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Supplier: ECACC (European Collection of Cell Cultures)
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver
Test concentrations with justification for top dose:
Experiment 1, 5-hour treatment period without S9 mix:
15, 17.5, 20, 22.5, 25, 27.5, 30 and 32.5 μg/mL
Experiment 1, 5-hour treatment period with S9 mix:
2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 μg/mL
Experiment 2, 20-hour treatment period without S9 mix:
12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 μg/mL
Experiment 2, 5-hour treatment period with S9 mix:
2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 μg/mL
Vehicle / solvent:
- Solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
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: 10^6 cells per dish

DURATION
- Exposure duration: 5 h with S9 mix
- Exposure duration: 20 h without S9 mix
- Expression time (cells in growth medium): 19 h
- Selection time: 8 d

SELECTION AGENT: hypoxanthine Ham's (F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
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,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
The test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a 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 historical negative control data (based 95% control limit).
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Statistical analysis was done with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- mutant frequency between the historical control and the test item or positive control item treated groups.

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. If the obtained result was positive, 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. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
with S9: 15, 17.5 and 20 µg/mL; without S9: 30 µg/mL
Vehicle controls validity:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES: yes
Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control and results were noted as percentage of cells in relation to the negative control. pH and osmolality were considered for dose level selection. Results of the Pre-test on cell toxicity were used for dose selection of the test item used in the Main Mutation Assays.

HISTORICAL CONTROL DATA
please refer to "any other information including tables"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control

Table 1 Main mutation assay summary of results with S9

 

ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABS.C.E.
(%)

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Negative Control a

199.3

±

1.53

100

100

0

0

1

0

3

4

99

4.04

 

Pos. Control
(
DMBA 20µg/mL) a

117.7

±

2.52

59

81

112

97

111

118

113

551

80

688.75**

 

TEST ITEM

 

2.5 µg/mL a

192.3

±

2.08

96

98

0

0

1

1

0

2

97

2.06

 

5 µg/mL a

179.3

±

5.13

90

98

1

1

0

0

1

3

97

3.09

 

7.5 µg/mL a

166.0

±

0.00

83

98

0

0

0

0

2

2

97

2.06

 

10 µg/mL a

84.7

±

2.52

42

97

0

0

0

2

1

3

96

3.13

 

12.5 µg/mL a

40.3

±

0.58

20

97

0

0

0

2

0

2

96

2.08

 

 

ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABS.C.E.
(%)

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Negative Control b

199.7

±

2.52

100

100

2

0

0

2

1

5

100

5.00

 

Pos. Control
(
DMBA 20µg/mL) b

118.0

±

4.00

59

78

109

108

101

105

107

530

78

679.49**

 

TEST ITEM

 

2.5 µg/mL b

189.0

±

1.00

95

99

0

0

1

1

0

2

99

2.02

 

5 µg/mL b

180.0

±

1.00

90

99

1

3

1

0

0

5

99

5.05

 

7.5 µg/mL b

164.0

±

1.00

82

99

0

0

1

1

0

2

99

2.02

 

10 µg/mL b

84.7

±

1.53

42

99

1

1

1

0

0

3

98

3.06

 

12.5 µg/mL b

38.0

±

1.00

19

97

0

1

0

1

0

2

97

2.06

 

a = parallel for mutation.

b = parallel for mutation.

ABS. C.E. =absolute cloning efficiency

DMBA=7.12-Dimethylbenzanthracene

FREQ = frequency

Pos. = positive

VEH. = DMSO

**= p < 0.01

Table 2 Main mutation assay summary of results without S9

 

ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABS.C.E.
(%)

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Negative Control a

199.3

±

0.58

100

100

0

0

0

2

3

5

101

4.95

 

Pos. Control a
(
EMS 1.0µL/mL) a

43.0

±

2.0

22

63

180

181

180

188

184

913

64

1426.56**

 

TEST ITEM

 

15 µg/mL a

196.7

±

1.53

99

100

0

0

0

1

1

2

100

2.00

 

17.5 µg/mL a

181.7

±

2.52

91

99

0

0

0

0

2

2

100

2.00

 

20 µg/mL a

175.7

±

2.08

88

99

0

0

0

0

2

2

100

2.00

 

22.5 µg/mL a

165.0

±

3.00

83

99

1

3

0

0

1

5

100

5.00

 

25 µg/mL a

143.7

±

3.51

72

99

2

0

0

0

0

2

100

2.00

 

27.5 µg/mL a

126.3

±

1.53

63

99

0

0

0

0

2

2

99

2.02

 

30 µg/mL a

65.0

±

1.73

33

97

0

1

3

1

0

5

98

5.10

 

32.5 µg/mL a

35.3

±

1.53

18

98

3

0

0

0

0

3

96

3.13

 

 

ACTIVATION
TEST
CONDITION

SURVIVAL TO TREATMENT

REL. POPU-
LATION
GROWTH (%)
OF CONTROL

MUTANT COLONIES
DISH NUMBER

TOTAL
MUTANT
COLONIES

ABS.C.E.
(%)

MUTANT
FREQ.
X 10-6

 

MEAN COLONY
NUMBER
S.D.

PERCENT
VEH. CONTROL

1

2

3

4

5

 

Negative Control b

199.3

±

2.08

100

100

0

1

0

1

2

4

101

3.96

 

Pos. Control a
(
EMS 1.0µL/mL) b

42.3

±

2.08

17

66

181

192

189

182

185

929

66

1407.58**

 

TEST ITEM

 

15 µg/mL b

197.3

±

1.53

99

99

0

1

2

0

0

3

100

3.00

 

17.5 µg/mL b

184.7

±

1.53

93

99

0

2

0

0

0

2

100

2.00

 

20 µg/mL b

175.3

±

1.53

88

100

0

0

0

2

0

2

101

1.98

 

22.5 µg/mL b

166.0

±

2.65

83

99

0

1

0

0

3

4

100

4.00

 

25 µg/mL b

145.0

±

3.00

73

98

1

1

1

0

0

3

99

3.03

 

27.5 µg/mL b

125.7

±

1.15

63

99

0

0

0

2

0

2

99

2.02

 

30 µg/mL b

67.3

±

2.52

34

99

1

1

0

0

1

3

98

3.06

 

32.5 µg/mL b

33.7

±

1.53

17

98

3

0

0

0

1

4

98

4.08

 

a = parallel for mutation.

b = parallel for mutation.

ABS. C.E. =absolute cloning efficiency

EMS =Ethyl methanesulfonate

FREQ = frequency

Pos. = positive

VEH. = DMSO

**= p < 0.01

Table 3 historical control data of solvent control

 

Without S9 mix

With S9 mix

5-hour treatment

20-hour treatment

5-hour treatment

Mean

3.98

3.99

4.09

SD

0.70

0.67

0.70

Lower confidence interval

2.46

2.53

2.57

Upper confidence interval

5.50

5.44

5.61

n

13

13

13

Table 4 historical control data of positive control

 

Without S9 mix

With S9 mix

5-hour treatment

20-hour treatment

5-hour treatment

Mean

1217.11

1229.36

610.24

SD

59.75

196.79

54.58

Lower confidence interval

1086.91

800.55

491.31

Upper confidence interval

1347.31

1658.17

729.18

n

13

13

13

Conclusions:
The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells. Thus, the test item was not mutagenic under the conditions of this study.
Executive summary:

The test item, dissolved in Dimethyl sulfoxide (DMSO), was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

Experiment 1, 5-hour treatment period without S9 mix:

15, 17.5, 20, 22.5, 25, 27.5, 30 and 32.5 μg/mL

Experiment 1, 5-hour treatment period with S9 mix:

2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 μg/mL

Experiment 2, 20-hour treatment period without S9 mix:

12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 μg/mL

Experiment 2, 5-hour treatment period with S9 mix:

2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)). Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted. The increases in mutation frequency in the positive control cultures with DMBA were a little bit above the historical control data range (779.73 and 735.53). However, these alterations did not influence the quality or integrity of the study. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (0.4 or 1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data. The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxicity. Thus, the test item was not mutagenic under the conditions of this study.

Genetic toxicity in vivo

Description of key information

The test substance (50% active ingredient) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Hence, the test substance was concluded to be negative in the micronucleus in vivo test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-12-21 and 2002-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals, 1996
Qualifier:
according to guideline
Guideline:
other: ICH Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals, 1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Drawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 27.6 - 31.5 g (male), 23.5 - 28.1 g (female)
- Housing: Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: certified laboratory rodent chow (Harlan TEKLAD certified Rodent 7012C); ad libitum
- Water: tap water (Washington Suburban Sanitary Commission, Potomac Plant); ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle: corn oil
- Justification for choice of solvent/vehicle: based on a solubility determination of the test article and compatibility of the vehicle with the test system
Details on exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered at a dose volume of 20 mL/kg bw.
Frequency of treatment:
single intraperitoneal injection
Remarks:
Doses / Concentrations:
300, 600 and 1200 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (50 mg/kg)
Tissues and cell types examined:
- Bone marrow cells were examined microscopically for micronucleated polychromatic erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose selection based on a pilot toxicity study followed by a toxicity study.

TREATMENT AND SAMPLING TIMES: Bone marrow cells were collected after 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenmald-Giemsa and permanently mounted.

METHOD OF ANALYSIS: Bone marrow cells, polychromatic and normochromatic erythrocytes were analyzed for the presence of micronuclei. For analysis slides were coded using a random number table. Using medium magnification (10x40), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (10x100), 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total reythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:
The test article was considered to induce positive results if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay is recommended. The test article is considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Criteria for a valid test - mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the positive control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group.
Statistics:
According to Kasten-Bowman Tables
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
no toxicity was observed up to 1200 mg/kg which is the highest dose used in the micronucleus study
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Pilot Toxicity Study: Mortality and clinical signs included prostation were observed in males and females at the highest dose of 2000 mg/kg. Lethargy and pilorection were observed in males at 100 and 1000 mg/kg and in males and females at 2000 mg/kg. Ataxia, paralysis and irregular brathing were noted in males and females at 2000 mg/kg.In addition, pilorection was observed in males at 10 mg/kg.

Toxicity Study: Mortality was observed at concentrations of 1400 mg/kg and higher. Different clinical signs included lethargy and pilorection were observed at all doses in males and females. The maximum tolerated dose was estimated to be 1200 mg/kg.

Definitive study - no mortality was observed.
Clinical signs including lethargy and piloerection were noted in 300, 600, and 1200 mg/kg dose groups. Slight reductions (up to 4%) in the ratio of polychromatic to total erythrocytes were observed in some test article-treated groups relative to the respective vehicle controls. No significant increase in micronucleated polychromatic erythrocytes (PCEs) were observed in any test substance treated group (0.7 or less per 1000 PCEs). A significant increase (p<0.05) in micronucleated polychromatic erythrocytes was observed in the positive control group (~ 20 per 1000 PCEs). The validity criteria were met.
Conclusions:
Interpretation of results: negative
The test substance (50% active ingredient) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Hence, the test substance was concluded to be negative in the micronucleus in vivo test.
Executive summary:

The test article (50% active ingredient) was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess toxicity of the test article and set dose levels for the definitive study consisted of a pilot study followed by a toxicity study. The second phase, the definitive micronucleus study, was designed to evaluate the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. In both phases of the study, test and control articles were administered at a dose volume of 20 mL/kg body weight by a single intraperitoneal injection.

The test article was soluble in corn oil at 200 mg/mL, the maximum concnetration tested in the solubility test. All test article dosing formulations were delivered to the test system as light yellow solutions.

Cyclophosphamide monhydrate (CP) was used as the positive control article at a dose of 50 mg/kg bw.

In the pilot toxicity study, male mice (2/group) were dosed with 1, 10, 100 or 1000 mg test article/kg body weight and male and female mice (5/sex/group) were dosed with 2000 mg test article/kg body weight. Mortality was observed in 4/5 males and 5/5 females at 2000 mg/kg bw. Clinical signs immediately following dose administration included: prostration in males and females at 2000 mg/kg bw. Lethargy and pilorection were observed in males at 100 and 1000 mg/kg bw and in males and females at 2000 mg/kg bw. Ataxia, paralysis and irregular breathing were noted in males and females at 2000 mg/kg bw. In addition, pilorection was observed in males at 10 mg/kg bw.

In the toxicity assay, male and female mice (5/sex/group) were dosed with 1200, 1400, 1600 or 1800 mg test article/kg body weight. Mortality was observed at concentrations of 1400 mg/kg bw and higher. Different clinical signs included lethargy and pilorection were observed at all doses in males and females. The maximum tolerated dose was estimated to be 1200 mg/kg bw.

In the definitive micronucleus study, male and female mice (5/sex/group) were dosed with 300, 600 or 1200 mg test article/kg body weight, vehicle or positive control article. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs following dose administration included: lethargy and pilorection in males and females at 300, 600 and 1200 mg/kg bw. Bone marrow cells were collected 24 and 48 hours after treatment. Slight reductions (up to 4%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some test article-treated groups relative to the respective vehicle controls. These reductions suggest that the test article did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman).

The validity critria for the positive control (Cyclophosphamide monohydrate) and the negative control (corn oil) were met.

It can be concluded that the test substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. The test substance was negative in the mouse micronucleus assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies

Ames test

The mutagenic potential of the test substance (50.1% purity) was tested in the Bacterial Reverse Mutation Assay in Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA. The test item was dissolved in acetone. In the Initial Mutation Test the following concentrations were examined: In absence of metabolic activation (-S9 Mix): 5000; 1600; 500; 160; 50 and 16 μg/plate; and in the presence of metabolic activation (+S9 Mix): 1000; 750; 500; 160; 50; 32 and 16 μg/plate. Due to the unequivocal demonstrative positive results obtained in the experiment I, (Plate Incorporation Test) the performance of an additional confirmatory experiment was considered as not necessary for the final conclusion of the study. The concentrations, including the controls, were tested in triplicate. In the performed experiment positive and negative (vehicle) controls were run concurrently. In the Initial Mutation Test following treatment with the test substance significant, biological relevant, dose-related changed revertant colony number increases, unequivocal positive results were noticed in the concentration range of 5000-160 μg/plate in S. typhimurium TA100, in the range of 5000-500 μg/plate in TA1535, and at 5000 and 1600 μg/plate in TA98, TA1537 and E. coli WP2 uvrA, in the absence of exogenous metabolic activation (-S9 Mix). Positive results were obtained at 750 and 500 μg/plate in E. coli WP2 uvrA, at 500 and 160 μg/plate in S. typhimurium TA100 and at 160 μg/plate in S. typhimurium TA98, with addition of metabolic activation (+S9 Mix). The mutagenic effect of the test item was unequivocal characteristic and very intensive. Signs of cytotoxicity (absent or reduced revertant colony numbers and/or affected background lawn development) were observed in all tested strains only in presence of metabolic activation. The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item (acting as direct mutagen and pro-mutagen, in absence and also in the presence of exogenous metabolic activation) induced gene mutations by frameshift and base-pair substitution in the genome of the tester strains used. Therefore, the test substance is considered mutagenic in this bacterial reverse mutation assay.

HPRT test

The test item, dissolved in Dimethyl sulfoxide (DMSO), was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item. Experiment 1, 5-hour treatment period without S9 mix: 15, 17.5, 20, 22.5, 25, 27.5, 30 and 32.5 μg/mL Experiment 1, 5-hour treatment period with S9 mix: 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 μg/mL Experiment 2, 20-hour treatment period without S9 mix: 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 μg/mL Experiment 2, 5-hour treatment period with S9 mix: 2.5, 5, 7.5, 10, 12.5, 15, 17.5 and 20 μg/mL In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)). Phenotypic expression was evaluated up to 8 days following exposure. In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted.The increases in mutation frequency in the positive control cultures with DMBA were a little bit above the historical control data range (779.73 and 735.53). However, these alterations did not influence the quality or integrity of the study.There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested. The validity of the test and the efficacy of the S9 mix were demonstrated by distinct and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with ethyl methanesulfonate (0.4 or 1.0 μL/mL) and 7,12-dimethyl benz[a]anthracene (20 μg/mL). The mutation frequency found in the positive controls was within the range of historical laboratory control data. The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to cytotoxicity. Thus, the test item was not mutagenic under the conditions of this study.

In vivo studies

Mammalian Erythrocyte Micronucleus Test

The test article (50% active ingredient) was tested in the mouse micronucleus assay. The assay was performed in two phases. The first phase (dose range-finding phase), designed to assess toxicity of the test article and set dose levels for the definitive study consisted of a pilot study followed by a toxicity study. The second phase, the definitive micronucleus study, was designed to evaluate the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female ICR mice. In both phases of the study, test and control articles were administered at a dose volume of 20 mL/kg body weight by a single intraperitoneal injection. The test article was soluble in corn oil at 200 mg/mL, the maximum concnetration tested in the solubility test. All test article dosing formulations were delivered to the test system as light yellow solutions. Cyclophosphamide monhydrate (CP) was used as the positive control article at a dose of 50 mg/kg bw. In the pilot toxicity study, male mice (2/group) were dosed with 1, 10, 100 or 1000 mg test article/kg body weight and male and female mice (5/sex/group) were dosed with 2000 mg test article/kg body weight. Mortality was observed in 4/5 males and 5/5 females at 2000 mg/kg bw. Clinical signs immediately following dose administration included: prostration in males and females at 2000 mg/kg bw. Lethargy and pilorection were observed in males at 100 and 1000 mg/kg bw and in males and females at 2000 mg/kg bw. Ataxia, paralysis and irregular breathing were noted in males and females at 2000 mg/kg bw. In addition, pilorection was observed in males at 10 mg/kg bw. In the toxicity assay, male and female mice (5/sex/group) were dosed with 1200, 1400, 1600 or 1800 mg test article/kg body weight. Mortality was observed at concentrations of 1400 mg/kg bw and higher. Different clinical signs included lethargy and pilorection were observed at all doses in males and females. The maximum tolerated dose was estimated to be 1200 mg/kg bw. In the definitive micronucleus study, male and female mice (5/sex/group) were dosed with 300, 600 or 1200 mg test article/kg body weight, vehicle or positive control article. No mortality was observed in any male or female mice in the micronucleus study. Clinical signs following dose administration included: lethargy and pilorection in males and females at 300, 600 and 1200 mg/kg bw. Bone marrow cells were collected 24 and 48 hours after treatment. Slight reductions (up to 4%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some test article-treated groups relative to the respective vehicle controls. These reductions suggest that the test article did not inhibit erythropoiesis. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control groups was observed in male or female mice at 24 or 48 hours after dose administration (p > 0.05, Kastenbaum-Bowman). The validity critria for the positive control (Cyclophosphamide monohydrate) and the negative control (corn oil) were met. It can be concluded that the test substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. The test substance was negative in the mouse micronucleus assay.

Conclusion

Tert-butyl peracetate is considered mutagenic in the absence and presence of exogenous metabolic activation in the bacterial reverse mutation assay (Ames test). However, the test item did not induce increases in mutant frequency in the in vitro HPRT test, when tested up to cytotoxic concentrations. The Micronucleus test in vivo also revealed negative results.

Kirkland et al. (Mutation Research 775-776 (2014) 69-80) conclude that:

“(...) in the case of an Ames-positive chemical, negative results in 2 in vitro mammalian cell tests covering both mutation and clastogenicity/aneugenicity endpoints should be considered as indicative of absence of in vivo genotoxic or carcinogenic potential."

This therefore implies for the mutagenicity endpoint a negative “In Vitro Mammalian Cell Gene Mutation Test” supported by in silico data is sufficient evidence to waive the need for in vivo gene mutation testing.

In this situation the Ames is positive, the HPRT is negative and the in vivo micronucleus is negative.

In silico methods indicate that the mutagenicity endpoint is negative:

Six different QSAR models (ACD TOXSUITE, TOPKAT, VEGA, DEREK, Danish QSAR Database,) are negative for (Ames) mutagenicity and or carinogenicity, with the substance falling in the applicability domain of the respective model. Please refer to Overview table attached to IUCLID Section 13. Single positive or inconclusive predictions by QSAR were only made where the substance does not fall in the applicability domain of the model.

In conclusion, based on the available information further in vivo mutagenicity testing is not required.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on the the available test results, the test item is not classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.