Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial: Ames study. Negative. OECD 471; Reliability = 1.

Mutagenic effects - bacterial: Ames study. Negative. OECD 471; Reliability = 1.

Mutagenic effects - bacterial: Ames study. Negative. OECD 471; Reliability = 1.

Mutagenic effects - bacterial: Ames study. Negative. OECD 471; Reliability = 1.

Clastogenic effects - mammalian: Chromosome aberrations in human peripheral blood lymphocytes. Negative in the absence of S9, positive in the presence of S9. OECD 473; Reliability = 1.

Clastogenic effects - mammalian: Chromosome aberrations in human peripheral blood lymphocytes. Positive in the absence of S9, negative in the presence of S9. OECD 473; Reliability = 1.

Clastogenic effects - mammalian: Chromosome aberrations in human peripheral blood lymphocytes. Negative. OECD 473; Reliability = 1.

Clastogenic effects - mammalian: Chromosome aberrations in human peripheral blood lymphocytes. Negative. OECD 473; Reliability = 1.

Mutagenic effects - mammalian: CJO/HGPRT assay. Negative. OECD 476; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-1 (2000)
Deviations:
no
GLP compliance:
yes
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:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Under the conditions of the assay, the test substance therefore, gave a negative, i.e., non-mutagenic response in S. typhimurium strains and E. coli strains in both the presence and absence of S9.
Executive summary:

The test substance was evaluated in a bacterial mutagenicity assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and one strain of Escherichia coli (WP2P uvrA) in the presence and absence of a rat liver - derived metabolic activation system (S9-mix), following protocols complying with OECD Guideline Numbers 471, U.S. EPA OPPTS Guideline 870.5100, EC Directive 440/2008/EC Method B.13/14, and the MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-1).

 

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revertant colonies in any of the tester strains used, either in the presence or absence of an auxiliary metabolizing system (S9).

 

In each experiment, the positive controls responded as expected indicating that the assay was performing satisfactorily.

 

Under the conditions of the assay, the test substance therefore, gave a negative, i.e., non-mutagenic response in S. typhimurium strains and E. coli strains in both the presence and absence of S9.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan 12-Nousan-8147 Guideline Number 2-1-19-1 (2000)
Deviations:
no
GLP compliance:
yes
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
Additional strain / cell type characteristics:
other: uvrA
Metabolic activation:
with and without
Metabolic activation system:
rat liver - derived metabolic activation system (S9-mix)
Test concentrations with justification for top dose:
Toxicity/mutagenicity experiment: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate; Confirmatory test: 50, 150, 500, 1500 and 5000 μg per plate
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Under the conditions of the assay, the test substance gave a negative, i.e., non-mutagenic response in S. typhimurium strains and E. coli strains in both the presence and absence of S9.
Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Test using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. Dosing formulations were adjusted to compensate for the purity of the test substance (98.5%), using a correction factor of 1.015 (OECD Section 4 (Part 471), Guideline for the Testing of Chemicals (1997); U.S. EPA OPPTS Guideline 870.5100 (1998); EC Directive 440/2008/EC Method B.13/14; and MAFF Japan 12-Nousan-8147 Guideline Number 2-1-19-1 (2000))

 

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. After sonication at 21.8ºC for 15 minutes in the solubility test, the test substance formed a clear solution in DMSO at a maximum concentration of approximately 400 mg/mL.

 

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Neither precipitate nor toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

 

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Neither precipitate nor toxicity was observed.

 

The results of the Bacterial Reverse Mutation Test indicate that, under the conditions of this study, the test substance did not exhibit any mutagenic responses in either the presence or absence of Aroclor-induced rat liver S9. Therefore, the test substance was concluded to be negative in this assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-1 (2000)
Deviations:
no
GLP compliance:
yes
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:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
In the toxicity-mutation test, 66.7, 100, 333, 667, 1000, 3000, 4000 and 5000 μg/plate and in the Confirmatory test: 667, 1000, 3000, 4000, and 5000 μg/plate
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 4-nitroquinoline N-oxide, acridine mutagen ICR-191, and 2-aminoanthracene
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.

A data set may be judged equivocal if there is a biologically relevant increased response that only partially meets criteria for a positive response. A response will be evaluated as negative if it is neither positive nor equivocal.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1 - Mutagenicity test in Salmonella typhimurium TA98 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

21

5

Positive Control*

306

46

    667

18

1

1000

26

4

3000

22

2

4000

37

7

5000

23

5

 

* 1.0 μg/plate 2-Nitrofluorene

 

Table 2 - Mutagenicity test in Salmonella typhimurium TA100 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

94

15

Positive Control*

1145

45

    667

78

19

1000

90

12

3000

71

5

4000

90

6

5000

83

17

 

* 2.0 μg/plate Sodium azide

 

Table 3 - Mutagenicity test in Salmonella typhimurium TA1535 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

12

3

Positive Control*

965

56

    667

13

6

1000

13

1

3000

12

3

4000

12

1

5000

12

3

 

* 2.0 μg/plate sodium azide

 

Table 4 - Mutagenicity test in Salmonella typhimurium TA1537 without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

9

3

Positive Control*

1321

148

    667

7

3

1000

10

4

3000

7

3

4000

7

3

5000

5

3

 

* 2.0 μg/plate ICR-191

 

Table 5 - Mutagenicity test in Escherichia coli WP2uvrA without S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

21

1

Positive Control*

698

76

    667

19

1

1000

17

5

3000

19

4

4000

18

5

5000

12

1

 

* 1.0 μg/plate 4-Nitroquinoline-N-oxide

 

Table 6 - Mutagenicity test in Salmonella typhimurium TA98 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

26

5

Positive Control*

473

12

    667

21

9

1000

34

8

3000

30

6

4000

24

2

5000

26

8

* 2.5 μg/plate Benzo(a)pyrene

 

Table 7 - Mutagenicity test in Salmonella typhimurium TA100 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

98

14

Positive Control*

1445

101

    667

88

5

1000

85

11

3000

95

3

4000

91

10

5000

86

8

 

* 2.5 μg/plate 2-Aminoanthracene

 

Table 8 - Mutagenicity test in Salmonella typhimurium TA1535 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

7

1

Positive Control*

147

10

    667

10

6

1000

12

5

3000

7

4

4000

10

4

5000

8

3

 

* 2.5 μg/plate 2-Aminoanthracene

 

Table 9 - Mutagenicity test in Salmonella typhimurium TA1537 with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

8

1

Positive Control*

76

5

    667

5

1

1000

8

2

3000

6

2

4000

6

2

5000

8

6

 

* 2.5 μg/plate 2-Aminoanthracene

 

Table 10 - Mutagenicity test in Escherichia coli WP2uvrA with S9

 

Dose (µg/plate)

Mean Revertants/plate

SD

Vehicle (DMSO)

19

5

Positive Control*

145

22

    667

19

9

1000

20

4

3000

24

4

4000

16

4

5000

18

7

 

* 25 μg/plate 2-Aminoanthracene

Conclusions:
Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.
Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) (OECD Section 4 (Part 471), Guideline for the Testing of Chemicals (1997); U.S. EPA OPPTS Guideline 870.5100 (1998); EC Directive 440/2008/EC Method B.13/14; and MAFF Japan 12-Nousan-8147 Guideline Number 2-1-19-1 (2000)).

 

The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

 

Dimethyl sulfoxide (DMSO) was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

 

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 66.7, 100, 333, 667, 1000, 3000, 4000 and 5000 μg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No appreciable toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 4000 μg/plate with tester strains TA98, TA1535, and TA1537 in the presence of S9 activation; and at 5000 μg/plate with tester strains TA100 and WP2uvrA in the presence of S9 activation and tester strains TA1535 and TA1537 in the absence of S9 activation.

 

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 667, 1000, 3000, 4000, and 5000 μg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation (see Tables 1 -10 in the "Any other information on results incl. tables" section for details). No appreciable toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 4000 μg/plate for all tester strains in both test condition with the exception of TA1535 without S9 activation where precipitation was observed only at 5000 μg/plate and for WP2uvrA without S9 activation where no test substance precipitation was observed.

 

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-1 (2000)
Deviations:
no
GLP compliance:
yes
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
Additional strain / cell type characteristics:
other: E. coli uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Toxicity-mutation test: 66.7, 100, 333, 667, 1000, 3000, 4000, and 5000 μg/plate
Confirmatory test: 667, 1000, 3000, 4000, and 5000 μg/plate
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: ICR-191 and 2-aminoanthracene
Evaluation criteria:
For Strains TA1535 and TA1537, data will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 3.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

For Strains TA98, TA100 and WP2uvrA, data sets will be judged positive if the increase in mean revertants at the highest numerical dose response is ≥ 2.0-fold the mean concurrent negative control value (vehicle control). This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

A data set may be judged equivocal if there is a biologically relevant increased response that only partially meets criteria for a positive response. A response will be evaluated as negative if it is neither positive nor equivocal.
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate at 3000 μg/plate or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitation at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate at 4000 and 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.
Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9) (OECD Guidelines for the Testing of Chemicals: Health Effects, No. 471, EC Directive 440/2008/EC Method B.13/14, U.S. EPA OPPTS Guideline 870.5100, and MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-1 (2000)).

 

The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

 

Dimethyl sulfoxide (DMSO) was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at 50 mg/mL, the highest stock concentration that was prepared for use on this study.

 

In the toxicity-mutation test, the maximum dose evaluated was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 66.7, 100, 333, 667, 1000, 3000, 4000, and 5000 μg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 3000 μg/plate with tester strain TA1537 in the presence of S9 activation and starting at 4000 μg/plate for all other tester strains both with and without S9 activation.

 

Based on the toxicity-mutation test, the maximum dose evaluated in the mutagenicity test was 5000 μg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 μL plating aliquot. The dose levels used in this test were 667, 1000, 3000, 4000, and 5000 μg/plate for all tester strains. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No toxicity was observed at any dose level with any tester strain in either the absence or presence of S9. Test substance precipitation was observed starting at 4000 μg/plate with tester strains TA98, TA100, TA1535, and TA1537 in the absence of S9 activation, and with tester strains TA1535 and TA1537 in the presence of S9 activation. Test substance precipitation was observed at 5000 μg/plate with TA98 and TA100 in the presence of S9 activation and with WP2uvrA both in the presence and absence of S9 activation.

 

All criteria for a valid study were met. Under the conditions of this study, the test substance showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Chromosome aberration assay in mammalian cells
Species / strain / cell type:
other: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the dose levels tested ranged from 0.398 to 3980 μg/mL (10 mM)

Based on these findings of the preliminary toxicity assay, the doses chosen for the chromosome aberration assay ranged from 250 to 3980 μg/mL for the non-activated and S9-activated 4-hour treatment groups, and from 100 to 1500 μg/mL for the non-activated 20-hour treatment group.
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
The test substance was considered to have induced a positive response if it induces a statistically significant and dose-dependent increase the frequency of aberrant metaphases (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result may be considered equivocal. If neither criterion is met, the results were considered to be negative.
Statistics:
Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.
Species / strain:
other: Human peripheral blood lymphocytes
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
For structural chromosome aberrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
At 1000 μg/mL or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Human peripheral blood lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
For numerical chromosome aberrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
At 1000 μg/mL or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Human peripheral blood lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
For the induction of structural or numerical chromosome aberrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
At 1000 μg/mL or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
The results of the In Vitro Mammalian Chromosome Aberration Test indicate that under the conditions of this study, the test substance was considered negative for the induction of structural or numerical chromosome aberrations in the absence of Aroclor-induced rat liver S9. The test substance was considered positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the presence of Aroclor-induced rat liver S9.
Executive summary:

The test substance was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assay was used to evaluate the clastogenic potential of the test substance. The dosing preparations were adjusted for test substance purity (99.4%), using a correction factor of 1.006 (OECD, Section 4 (Part 473) (adopted 1997), U.S. EPA, OPPTS 870.5375: In Vitro Mammalian Chromosome Aberration Test (1998), EC, Directive 440/2008/EC Method B.10: Mutagenicity (2008), and MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000).

 

Dimethyl sulfoxide (DMSO) was used as the solvent based on the ability of the test substance to form workable suspensions in DMSO and compatibility with the target cells. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

 

In the preliminary toxicity assay, the dose levels tested ranged from 0.398 to 3980 μg/mL (10 mM). Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. Visible precipitate was observed in treatment medium at 3980 μg/mL, while dose levels ≤ 1194 μg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period for all test conditions. At the conclusion of the treatment period, hemolysis was observed at 3980 μg/mL in all three treatment groups. The osmolality and pH in treatment medium of the highest dose level tested, 3980 μg/mL, were within acceptable ranges. Substantial toxicity (at least 50% reduction in mitotic index relative to the solvent control) was observed at 3980 μg/mL in the non-activated and S9-activated 4-hour treatment groups, and at dose levels ≥ 1194 μg/mL in the non-activated 20-hour treatment group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 250 to 3980 μg/mL for the non-activated and S9-activated 4-hour treatment groups, and from 100 to 1500 μg/mL for the non-activated 20-hour treatment group.

 

In the initial chromosome aberration assay, the cells were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. All cells were harvested 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at dose levels ≥ 1250 μg/mL, while dose levels ≤ 1000 μg/mL were soluble in treatment medium at the beginning and conclusion of the treatment period. At the conclusion of the treatment period, hemolysis was observed at dose levels ≥ 2500 μg/mL in the non-activated and S9-activated 4-hour treatment groups. The highest dose selected for microscopic analysis of chromosome aberrations in each treatment condition, produced a greater than 50% reduction in mitotic index relative to solvent control. Two additional lower doses were included in the analysis.

 

The percentage of cells with structural aberrations in the non-activated 4 and 20-hour treatment groups was not significantly increased relative to the solvent control at any dose level evaluated (p > 0.05, Fisher’s Exact test). The percentage of cells with structural aberrations in the S9 activated 4-hour treatment group was statistically increased (3.5%) relative to the solvent control at the highest dose level evaluated (p ≤ 0.01, Fisher’s Exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05). The percentage of cells with numerical aberrations in the test substance-treated groups was not significantly increased relative to the solvent control at any dose level (p > 0.05, Fisher’s Exact test).

 

In order to confirm the significant increase in structural aberrations in the S9-activated 4-hour exposure group, the chromosome aberration assay was repeated (first repeat). Since the data generated did not confirm the original results, the assay was repeated again in the S9-activated 4-hour exposure group (second repeat). Data generated during the conduct of the first repeat assay are maintained in the study file, but not reported.

 

In the second repeat chromosome aberration assay, the dose levels tested were 500, 1000, 1250, 1500, 1700, 1850, 2000, 2200, 2350, 2500, 2600, 2700, and 2800 μg/mL. HPBL cells were treated in the presence of an Aroclor-induced S9 activation system for 4 hours. All cells were harvested 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at dose levels ≥ 1000 μg/mL, while dose level 500 μg/mL was soluble in treatment medium at the beginning and conclusion of the treatment period. The highest dose selected for microscopic analysis produced a greater than 50% reduction in mitotic index relative to solvent control and had visible precipitate at the end of treatment. Four additional lower doses were included in the analysis.

 

In the second repeat chromosome aberration assay, the percentage of cells with structural aberrations in the S9-activated 4-hour treatment group was statistically increased (2.5% and 4.0%) relative to the solvent control at 2000 and 2800 μg/mL, respectively (p ≤ 0.05 or p ≤ 0.01, Fisher’s Exact test). The Cochran-Armitage test was also positive for a dose response (p ≤ 0.05). The percentage of cells with numerical aberrations in the test substance-treated group was not significantly increased relative to the solvent control at any dose level (p > 0.05, Fisher’s Exact test).

 

The positive and solvent controls fulfilled the requirements for a valid test.

 

The results of the In Vitro Mammalian Chromosome Aberration Test indicate that under the conditions of this study, the test substance was considered negative for the induction of structural or numerical chromosome aberrations in the absence of Aroclor-induced rat liver S9. The test substance was considered positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the presence of Aroclor-induced rat liver S9.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In Vitro Mammalian Chromosome Aberration Test in Human Peripheral Blood Lymphocytes
Species / strain / cell type:
other: Human Peripheral Blood Lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced S9 activation system
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the dose levels tested were 0.398, 1.194, 3.98, 11.94, 39.8, 119.4, 398, 1194, and to 3980 μg/mL

Doses chosen for the chromosome aberration assay were 250, 500, and 1500 μg/mL for the 4-hour activated and non-activated treatment groups and 250, 500, and 800 μg/mL for the 20-hour non-activated treatment group.
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
Toxicity induced by treatment was based upon inhibition of mitosis and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.

The test substance was considered positive if it induced a statistically significant and dose-dependent increase the frequency of aberrant metaphases (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result were considered equivocal. If neither criterion was met, the results were considered to be negative.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison\ to historical control values, biological significance, etc.). In such cases, the Study Director will use sound scientific judgment and clearly report and describe any such considerations.
Statistics:
Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose responsiveness.
Species / strain:
other: Peripheral human blood lymphocytes
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
For the induction of structural chromosome aberrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
At 1000 μg/mL or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Peripheral human blood lymphocytes
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
For the induction of numerical chromosome aberrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
At 1000 μg/mL or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: Peripheral human blood lymphocytes
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
Negative for the induction of structural and numerical chromosome aberrations
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
At 1000 μg/mL or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
The results of the In Vitro Mammalian Chromosome Aberration Test in human peripheral blood lymphocytes indicate that under the conditions of this study, the test substance was concluded to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the non-activated test system. The test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in the S9-activated test system. The test substance was concluded to be positive in this in vitro test system.
Executive summary:

The test substance was tested in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system. A preliminary toxicity test was performed to establish the dose range for testing in the cytogenetic test. The chromosome aberration assays were used to evaluate the clastogenic potential of the test substance. Dose formulations were adjusted to compensate for the purity of the test substance (98.5%), using a correction factor of 1.015 (OECD, Section 4 (Part 473) (adopted 1997), U.S. EPA, OPPTS 870.5375: In Vitro Mammalian Chromosome Aberration Test (1998), EC, Directive 440/2008/EC Method B.10: Mutagenicity (2008), and MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000)).

 

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells. In the solubility test conducted at BioReliance, upon sonication for 15 minutes at 21.8ºC, the test substance formed a clear solution in DMSO at a maximum concentration of approximately 400 mg/mL. Cyclophosphamide and mitomycin C were evaluated as the concurrent positive controls for treatments with and without S9, respectively.

 

In the preliminary toxicity assay, the dose levels tested ranged from 0.398 to 3980 μg/mL (10 mM). Human peripheral blood lymphocytes were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. Visible precipitate was observed in treatment medium at 3980 μg/mL, while dose levels ≤ 1194 μg/mL were soluble in treatment medium at the beginning of the treatment period for all test conditions. At the conclusion of the treatment period, visible precipitate was observed in treatment medium at dose levels ≥ 1194 μg/mL, while dose levels ≤ 398 μg/mL were soluble in treatment medium for all test conditions. Also, at the conclusion of the treatment period, hemolysis was observed at 3980 μg/mL in all treatment conditions. Substantial toxicity (at least 50% reduction in mitotic index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Substantial toxicity was observed at 3980 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 1194 μg/mL in the non-activated 20-hour exposure group. Based on these findings, the doses chosen for the chromosome aberration assay ranged from 100 to 1500 μg/mL for all three treatment conditions.

 

In the chromosome aberration assay, the cells were treated in the absence and presence of an Aroclor-induced S9 activation system for 4 hours, and continuously for 20 hours in the absence of S9 activation. All cells were harvested 20 hours after treatment initiation. Visible precipitate was observed in treatment medium at dose levels ≥ 1000 μg/mL, while dose levels ≤ 800 μg/mL were soluble in treatment medium at the beginning of the treatment period for all test conditions. At the conclusion of the treatment period, in the non-activated and S9-activated 4-hour treatment groups, visible precipitate was observed in treatment medium at dose levels ≥ 1000 μg/mL, while dose levels ≤ 800 μg/mL were soluble in treatment medium. In the non-activated 20-hour exposure group, visible precipitate was observed in treatment medium at 1500 μg/mL, while dose levels ≤ 1000 μg/mL were soluble in treatment medium at the conclusion of the treatment period.

 

Substantial toxicity was not observed at any dose level in the non-activated 4-hour exposure group. Substantial toxicity was observed at 1500 μg/mL in the S9-activated 4-hour exposure group, and at dose levels ≥ 800 μg/mL in the non-activated 20-hour exposure group. Based on these findings and upon consultation with the Sponsor, the doses chosen for microscopic analysis

were 250, 500, and 1500 μg/mL for the non-activated and S9-activated 4-hour treatment groups, and 250, 500, and 800 μg/mL for the non-activated 20-hour treatment group.

 

The percentage of cells with structural aberrations in the non-activated 4-hour exposure group was statistically increased (3.5%) relative to vehicle control at 1500 μg/mL (p ≤ 0.01, Fisher's Exact test). The percentage of cells with structural aberrations in the non-activated 20-hour exposure group was statistically increased (17.0%) relative to vehicle control at 800 μg/mL (p ≤ 0.01, Fisher's Exact test). The percentage of cells with structural aberrations in the S9-activated 4-hour exposure group was not significantly increased relative to vehicle control at any dose level (p > 0.01, Fisher's Exact test). The percentage of cells with numerical aberrations in the test substance-treated groups was not significantly increased relative to vehicle control at any

dose level (p > 0.01, Fisher's Exact test). The positive and vehicle controls fulfilled the requirements for a valid test.

 

The results of the In Vitro Mammalian Chromosome Aberration Test in human peripheral blood lymphocytes indicate that under the conditions of this study, the test substance was concluded to be positive for the induction of structural and negative for the induction of numerical chromosome aberrations in the non-activated test system. The test substance was concluded to be negative for the induction of structural and numerical chromosome aberrations in the S9-activated test system. The test substance was concluded to be positive in this in vitro test system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Chromosome aberration assay in mammalian cells
Species / strain / cell type:
other: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
In the initial chromsome aberration assay, concentrations were 5, 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750 and 2000 μg/mL;
In the repeat chromsome aberration assay, concentrations were 50, 100, 250, 500, 750, 850 950, 1250, 1500 and 2000 μg/mL;
In the 4-hour cytogenetic non-activated assay, concentrations were 250, 750, and 950 μg/mL;
In the 4-hour cytogenetic activated assay, concentrations were 250, 500, and 950 μg/mL; and in the 22-hour cytogenetic assay, concentrations were 100, 250, and 750 μg/mL, selected based on toxicity
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
The following conditions were used as a guide to determine a positive response:

• A statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response.
• The observed increased frequencies were accompanied by a concentration-related increase when evaluated by the trend test.
• Any of the results were outside the 95% control limit distribution of the historical negative control data.
• Note: Statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.

The following condition was used as a guide to determine an equivocal response:

• Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-responsive trend.

The test substance was judged negative if the following conditions were met:

• There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group.
• There was no concentration-related increase when evaluated with an appropriate trend test.
• All results were within the 95% control limit of the distribution of the historical negative control database.
Statistics:
The clastogenic potential of the test substance was assessed based on its ability to induce structural chromosome aberrations. The experimental unit is the cell; therefore the percentage of cells with structural aberrations was used for the assessment.

Data were evaluated using scientific judgment. Statistical analysis was used as a guide to determine whether or not the test substance induced a positive response. Interpretation of the statistical analysis also relied on additional considerations including the magnitude of the observed test substance response relative to the vehicle control response and the presence of a dose-responsive trend. Statistical analysis consisted of a Fisher’s exact test (with Bonferroni-Holm Adjustment) to compare the percentage of cells with structural or numerical aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the vehicle control response. A Cochran-Armitage test for dose responsiveness was conducted only on values within a test condition only if statistically significant values, based on the Fisher’s exact test, are found. At the discretion of the study director, statistical analyses may be conducted on the percentage of cells with numerical aberrations as well.
Key result
Species / strain:
other: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
For structural or numerical chromosome aberrations
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 1250 μg/mL in the 4-hour non-activated assay, at 950 μg/mL in the 4-hour activated assay, and at 750 μg/mL in the 22-hour non-activated assay
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1 - Chromosome aberration assay – cytogenetic analysis of human peripheral blood lymphocytes treated with the test substance in the absence of exogenous metabolic activation (4-hour treatment, 22-hour harvest)

 

Treatment (μg/mL)

Slide

Mitotic Index (%)

Numerical cells scored

Structural cells scored

Aberrant numerical cells

Aberrant Structural cells

Average Aberrations per cell

DMSO vehicle

A

B

12.8

13.6

150

150

150

150

0

0

1

3

0.007

0.020

250

A

B

12.0

13.2

150

150

150

150

0

0

3

2

0.020

0.013

750

A

B

12.0

15.2

150

150

150

150

1

0

2

1

0.013

0.007

950

A

B

8.2

7.2

150

150

150

150

0

0

3

3

0.020

0.020

Positive Control*

A

B

6.6

7.8

50

50

50

50

0

0

23

16

0.460

0.320

 

*Mitomycin-C 0.4μg/mL

 

Table 2 - Chromosome aberration assay – cytogenetic analysis of human peripheral blood lymphocytes treated with the test substance in the presence of exogenous metabolic activation (4-hour treatment, 22-hour harvest)

 

Treatment (μg/mL)

Slide

Mitotic Index (%)

Numerical cells scored

Structural cells scored

Aberrant numerical cells

Aberrant Structural cells

Average Aberrations per cell

DMSO vehicle

A

B

8.2

12.6

150

150

150

150

0

0

2

3

0.013

0.020

250

A

B

12.2

14.0

150

150

150

150

0

0

0

2

0.000

0.013

500

A

B

10.4

8.8

150

150

150

150

0

0

3

3

0.020

0.020

950

A

B

4.0

3.8

150

150

150

150

0

0

1

0

0.007

0.000

Positive Control*

A

B

10.4

11.8

150

50

150

50

0

0

14

15

0.093

0.300

 

* Cyclophosphamide

 

Table 3 - Chromosome aberration assay – cytogenetic analysis of human peripheral blood lymphocytes treated with the test substance in the absence of exogenous metabolic activation (22-hour continuous treatment)

 

Treatment (μg/mL)

Slide

Mitotic Index (%)

Numerical cells scored

Structural cells scored

Aberrant numerical cells

Aberrant Structural cells

Average Aberrations per cell

DMSO vehicle

A

B

13.4

10.2

150

150

150

150

0

0

4

1

0.027

0.007

250

A

B

8.8

6.2

150

150

150

150

0

0

3

2

0.020

0.013

750

A

B

8.0

7.6

150

150

150

150

0

0

3

1

0.020

0.007

950

A

B

6.0

4.6

150

150

150

150

0

0

2

1

0.013

0.007

Positive Control*

A

B

6.0

6.8

50

50

50

50

0

0

15

21

0.300

0.420

 

* MMC 0.2 μg/mL

Conclusions:
All criteria for a valid study were met. Under the conditions of this study, the test substance was not found to induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in the non-activated and S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.
Executive summary:

The test substance was evaluated for its ability to induce structural chromosome aberrations in vitro using human peripheral blood lymphocytes in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). Numerical aberrations were also recorded (OECD, Section 4 (Part 473) (adopted 1997), U.S. EPA, OPPTS 870.5375: In Vitro Mammalian Chromosome Aberration Test (1998), EC, Directive 440/2008/EC Method B.10: Mutagenicity (2008), and MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000)).

 

The test substance was formulated in dimethyl sulfoxide (DMSO) and was a clear dark amber/yellow solution at 200 mg/mL, the highest stock concentration prepared for the chromosome aberration assay.

 

The concentrations chosen for all test conditions in the chromosome aberration assay were 5, 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750 and 2000 μg/mL. Test substance precipitation was observed at concentrations ≥500 μg/mL at the beginning of treatment in all test conditions, and at concentrations ≥1000 μg/mL at the end of treatment for all test conditions. Due to low and variable mitotic index results the assay was repeated.

 

The concentrations chosen for all test conditions in the repeat assay were 50, 100, 250, 500, 750, 850 950, 1250, 1500 and 2000 μg/mL. Test substance precipitation was observed at concentrations ≥750 μg/mL at the beginning of treatment and at ≥950 μg/mL at the end of treatment for all test conditions. Substantial toxicity (55±5% mitotic reduction in relation to the vehicle control) was observed in the 4-hour non-activated test condition starting at 1250 μg/mL and in the 22-hour non-activated test condition starting at 750 μg/mL. Toxicity of 62.5% was observed in the 4-hour activated test condition at 950 μg/mL.

Cytogenetic evaluations were conducted at 250, 750 and 950 μg/mL in the 4-hour non-activated condition. These concentration levels were selected based on the lowest precipitating concentration at the end of treatment period. In the 4-hour activated test condition, cytogenetic evaluations were conducted at 250, 500 and 950 μg/mL, selected based on toxicity. In the 22-hour test condition, cytogenetic evaluations were conducted at 100, 250 and 750 μg/mL, selected based on toxicity.

 

The percentage of cells with structural and numerical aberrations in the test substance-treated groups was not significantly increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher’s exact test).

 

All criteria for a valid study were met. Under the conditions of this study, the test substance was not found to induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in the non-activated and S9-activated test systems. It was concluded that the test substance was negative in this in vitro test (see Tables 1 -3 in the "Any other information on results incl. tables" section for details).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In Vitro Mammalian Chromosome Aberration Test in Human Peripheral Blood Lymphocytes
Species / strain / cell type:
other: Human peripheral blood lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Initial chromosome aberration assay: 5, 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750 and 2000 μg/mL
Repeat chromosome aberration assay: 250, 500, 550, 600, 700 and 750 μg/mL
Confirmatory chromosome aberration assay: 250, 500, 550, 600, 700, 750, 850, 950, 1250 and 2000 μg/mL
Cytogenetic evaluations were conducted at 250, 500 and 750 μg/mL in the 4- and 22-hour non-activated test conditions
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Evaluation criteria:
The following conditions were used as a guide to determine a positive response:

• A statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with structural aberrations was seen in one or more treatment groups relative to the vehicle control response.
• The observed increased frequencies were accompanied by a concentration-related increase when evaluated by the trend test.
• Any of the results were outside the 95% control limit distribution of the historical negative control data.
• Note: Statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.

The following condition was used as a guide to determine an equivocal response:

• Results observed in any of the assays resulted in statistically significant elevations in structural chromosome aberrations at more than one test concentration level, except the highest dose, without demonstrating a dose-responsive trend.

The test substance was judged negative if the following conditions were met:

• There was no statistically significant increase in the percentage of cells with structural aberrations in any treatment group relative to the vehicle control group.
• There was no concentration-related increase when evaluated with an appropriate trend test.
• All results were within the 95% control limit of the distribution of the historical negative control database.
Statistics:
The clastogenic potential of the test substance was assessed based on its ability to induce structural chromosome aberrations. The experimental unit is the cell; therefore the percentage of cells with structural aberrations was used for the assessment.

Data were evaluated using scientific judgment. Statistical analysis was used as a guide to determine whether or not the test substance induced a positive response. Interpretation of the statistical analysis also relied on additional considerations including the magnitude of the observed test substance response relative to the vehicle control response and the presence of a dose-responsive trend. Statistical analysis consisted of a Fisher’s exact test (with Bonferroni-Holm Adjustment) to compare the percentage of cells with structural or numerical aberrations (or the percentage of cells with more than one aberration, if required) in the test substance treated groups with the vehicle control response. A Cochran-Armitage test for dose responsiveness was conducted only on values within a test condition only if statistically significant values, based on the Fisher’s exact test, are found. At the discretion of the study director, statistical analyses may be conducted on the percentage of cells with numerical aberrations as well.
Species / strain:
other: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 500 μg/mL or more
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
All criteria for a valid study were met. Under the conditions of this study, the test substance was not found to induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in the non-activated and S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.
Executive summary:

The test substance was evaluated for its ability to induce structural chromosome aberrations in vitro using human peripheral blood lymphocytes (HPBL) in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). Numerical aberrations were also recorded (OECD, Section 4 (Part 473) (adopted 1997), U.S. EPA, OPPTS 870.5375: In Vitro Mammalian Chromosome Aberration Test (1998), EC, Directive 440/2008/EC Method B.10: Mutagenicity (2008), and MAFF Japan, 12-Nousan-8147: Guideline Number 2-1-19-2 (2000)).

 

The test substance was formulated in dimethyl sulfoxide (DMSO), which formed a solution at 200 mg/mL, the highest stock concentration prepared for the assay.

 

The concentrations chosen for the chromosome aberration assay were 5, 10, 50, 100, 250, 500, 750, 1000, 1250, 1500, 1750 and 2000 μg/mL. Test substance precipitation was observed at ≥500 μg/mL in the beginning of treatment in all test conditions. At the end of treatment, precipitation was observed at ≥750 μg/mL in the 4-hour treatment conditions, and ≥1250 μg/mL in the 22-hour treatment condition. In the 4-hour non-activated test condition, the top concentration (750 μg/mL) selected for cytogenetic analysis was based on test substance precipitation at the end of treatment. In the 22-hour test condition, the top concentration selected for cytogenetic analysis (750 μg/mL) was based on substantial toxicity (55±5% mitotic reduction in relation to the vehicle control). In the 4-hour S9-activated test condition, the lowest precipitating concentration (750 μg/mL) could not be analyzed due to excessive toxicity; therefore, this test condition was repeated.

 

The concentrations chosen for the repeat assay for the 4-hour S9-activated test condition were 250, 500, 550, 600, 700 and 750 μg/mL. Test substance precipitation was observed at ≥550 μg/mL in the beginning of treatment. No test substance precipitation was observed at the end of treatment. To ensure a valid assay, a confirmatory assay for the 4-hour S9-activated test condition was conducted. The concentrations chosen for the confirmatory assay were 250, 500, 550, 600, 700, 750, 850, 950, 1250 and 2000 μg/mL. Test substance precipitation was observed at ≥500 μg/mL in the beginning of treatment and at 2000 μg/mL in the end of treatment. Substantial toxicity was observed starting at 500 μg/mL.

 

Cytogenetic evaluations were conducted at 250, 500 and 750 μg/mL in the 4- and 22-hour non-activated test conditions. In the 4-hour activated test condition, due to the variable cytotoxicity observed, cytogenetic evaluations were conducted at 250, 550 and 750 μg/mL from the main and confirmatory assays.

 

The percentage of cells with structural and numerical aberrations in the test substance-treated groups was not significantly increased above that of the vehicle control at any concentration (p ≥ 0.05, Fisher’s exact test).

 

All criteria for a valid study were met. Under the conditions of this study, the test substance was not found to induce structural or numerical chromosome aberrations in the in vitro mammalian chromosome aberration test in human peripheral blood lymphocytes in the non-activated and S9-activated test systems. It was concluded that the test substance was negative in this in vitro test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan 59-Nousan-4200, Testing Guidelines for Toxicity Studies (1985)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: CHO/HGPRT assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
HGPRT Locus
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
250, 500, 750, 1000, and 1500 μg/mL
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Evaluation criteria:
All conclusions are based on scientific judgement; however, the following will be used as a guide to interpretation of the data. The test substance will be considered to induce a positive response if there is a concentration-related increase in mutant frequencies with at least two consecutive doses showing mutant frequencies of > 40 mutants per 10(6) clonable cells. If a single point above 40 mutants per 10(6) clonable cells is observed at the highest dose, the results will be considered equivocal. If no culture exhibits a mutant frequency of > 40 mutants per 10(6) clonable cells, the test substance will be considered negative.
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
HGPRT locus
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitate at 1500 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
All criteria for a valid study were met. Under the conditions of this study, the test substance did not cause a positive response in the non-activated or S9-activated systems in the CHO/HGPRT Mutation Assay. Therefore, the test substance was concluded to be negative in this assay.
Executive summary:

The purpose of this screening study was to evaluate the mutagenic potential of the test substance based on quantitation of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus of Chinese hamster ovary (CHO) cells (OECD Guideline for the Testing of Chemicals, Guideline 476, EC Directive 440/2008/EC Method B.17,   US EPA Health Effects Test Guidelines, OPPTS 870.5300, and MAFF Japan 59-Nousan-4200, Testing Guidelines for Toxicity Studies).

The test substance was tested in the CHO/HGPRT Mutation Assay in the absence and presence of Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the dose range for the mutagenesis assay. The mutagenesis assay was used to evaluate the mutagenic potential of the test substance. Dosing formulations were adjusted to compensate for the purity (99.4%) of the test substance, using a correction factor of 1.006.

 

Dimethyl sulfoxide (DMSO) was selected as the solvent of choice based on the solubility of the test substance and compatibility with the target cells. After sonication at 36.5ºC for 20 minutes in the solubility test, the test substance formed a clear solution in DMSO at approximately 150 mg/mL.

 

In the preliminary toxicity assay, the maximum concentration of the test substance was 1500 μg/mL with and without S9 activation. There was visible precipitate in the treatment medium at 1500 μg/mL at the beginning and end of treatment. No substantial toxicity, i.e., cloning efficiency <50% of the solvent control, was observed at any concentration with or without S9 activation. Based on these findings, the concentrations chosen for the mutagenesis assay ranged from 250 to 1500 μg/mL for both the non-activated and S9-activated cultures.

 

In the mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10(6) clonable cells, were observed. There was visible precipitate in the treatment medium at 1500 μg/mL at the beginning and end of treatment. No toxicity, i.e., cloning efficiency <50% of the solvent control, was observed at any concentration with or without S9 activation.

 

All criteria for a valid study were met. Under the conditions of this study, the test substance did not cause a positive response in the non-activated or S9-activated systems in the CHO/HGPRT Mutation Assay. Therefore, the test substance was concluded to be negative in this assay.

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

Genetic toxicity in vivo

Description of key information

Clastogenic effects - mammalian: in vivo mouse micronucleus study; Negative. OECD 474; Reliability = 1

Clastogenic effects - mammalian: in vivo mouse micronucleus study; Negative. OECD 474; Reliability = 1

Clastogenic effects - mammalian: in vivo mouse micronucleus study; Negative. OECD 474; Reliability = 1

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In Vivo Micronucleus Test in Mice
Species:
mouse
Strain:
other: Crl: ICR (CD-I)
Details on species / strain selection:
The mouse has been routinely used as an animal model of choice for the mammalian bone marrow erythrocyte micronucleus assay. This strain is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test substances.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Mice were obtained from Charles River Laboratories at Stone Ridge, NY for dose range finding [DRF] assay and from Portage, MI for the definitive study.
- Age at study initiation: 7 weeks old
- Weight at study initiation: At DRF: 27.5-29.4 g [males] and 22.2-25.4 g [females] and for the definitive study: 27.8-34.1 g [males] and 23.3-28.3 g [females]
- Assigned to test groups randomly: yes
- Fasting period before study: No data
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage; Heat-treated Sani-Chip hardwood chips were used for bedding to absorb liquids
- Diet (e.g. ad libitum): Harlan 2018C Certified Global Rodent Diet
- Water (e.g. ad libitum): Tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 ± 3°F
- Humidity (%): 50 ± 20%
- Air changes (per hr): 10 changes of fresh HEPA-filtered air every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
0.1% Tween-80 in aqueous methylcellulose (0.5%)
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
No
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
DRF study: 3 male and 3 females
Definitive study: 5 males and 5 females in the control and 500 and 1000 mg/kg groups and 10 males and 10 females in the 2000 mg/kg group
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide monohydrate
Tissues and cell types examined:
micronucleated polychromatic erythrocytes (MPCEs) in the bone marrow
Evaluation criteria:
The test substance is considered to be positive if it induces a significant increase in MNPCE frequency (p ≤ 0.05) at any dose level or sampling time compared to the concurrent vehicle control.

The test substance is considered to be negative if no significant increase in MNPCE frequency is observed (p > 0.05) compared to the concurrent vehicle control.

Other criteria may be used in reaching a conclusion about the study results (e.g., magnitude of any increase, dose-dependency, comparison to historical control values, biological significance, etc.). Biological relevance of the results are considered first. Statistical methods may be used as an aid in evaluating the test results. Statistical significance is not the only determining factor for a positive response. Equivocal results
are clarified by further testing preferably using a modification of experimental conditions. The Study Director uses sound scientific judgment to clearly report and describe any such considerations.

Criteria for a Valid Test
• The MNPCE frequency of the vehicle controls must be within the historical vehicle control range, and the positive control must induce a significant increase (p ≤ 0.05, Kastenbaum-Bowman Tables) in MNPCE frequency as compared to concurrent vehicle control.
• Five animals/sex/group are available for analysis.
Statistics:
The frequency of MNPCEs and the proportion of PCEs to total erythrocytes were determined for each animal and treatment group. Statistical significance (p ≤ 0.05) was determined using the binomial distribution (Kastenbaum-Bowman tables).
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The following results were generated during the conduct of the definitive assay:

• No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and in the test substance treated groups appeared normal during the study period.

• No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio) in the male and female test substance groups relative to the respective vehicle control groups were observed at 24 or 48 hours post-dose, suggesting that the test substance did not induce cytotoxicity.

• No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance 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, binomial distribution, Kastenbaum-Bowman Tables).

• Cyclophosphamide monohydrate (CP), the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs
(p ≤ 0.05, binomial distribution, Kastenbaum-Bowman Tables) in both male and female mice.

• The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.
Conclusions:
The test substance was concluded to be negative in the mouse micronucleus assay.
Executive summary:

The test substance was evaluated for its genotoxic potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes (MPCEs) in bone marrow of male and female in Crl:CD-1 (CD-1) mice (OECD Guidelines for the Testing of Chemicals No 474; EPA OPPTS Guideline 870.5395; EC Directive 2008/32/EC Method B.12; and MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3).

 

The test substance vehicle, 0.1% Tween-80 in aqueous methylcellulose (4000 cP, 0.5%), was selected by the Sponsor based on good workability of the test substance in the vehicle and compatibility of the vehicle with the test system and route of administration. The vehicle was also used as the negative (vehicle) control and Cyclophosphamide monohydrate, at a dose of 40 mg/kg, was used as the positive control substance. The amount of test substance used in preparing each concentration was adjusted for purity using a correction factor of 1.006. All formulations were administered at a dose volume of 20 mL/kg body weight by a single oral gavage.

 

The Micronucleus test was conducted in two phases: a dose range finding (DRF) assay that determined the toxicity of the test substance and definitive phase (micronucleus study/assay), that determined the potential of the test substance to increase the incidence of MPCEs in the target bone marrow.

 

In the DRF assay, three male mice were initially exposed to the test substance at 1000, 1500 or 2000 mg/kg. In absence of mortality in the DRF assay, a dose of 2000 mg/kg was tested as high dose in the micronucleus assay. Two lower doses, one fourth and one half of the high dose, at 500 and 1000 mg/kg were also tested. The vehicle and positive control substances were run concurrently. Bone marrow cells [polychromatic erythrocytes (PCEs)] collected from all groups at 24 hours post–dose and from the vehicle and 2000 mg/kg treatment groups at 48 hour post-dose were examined microscopically for the presence of micronuclei (MNPCEs). In addition, the ratio of polychromatic erythrocytes to total erythrocytes was evaluated in the test substance groups relative to the respective vehicle control groups to reflect the test substance’s cytotoxicity.

 

In the DRF assay, no mortality was observed in any of the treatment groups. Both male and female mice in all treatment groups appeared normal during the study period. No appreciable changes to the mean group body weights were observed in any of the treatment groups.

 

The following results were generated during the conduct of the definitive assay:

 

• No mortality was observed in any of the treatment groups. All mice in the control substance (vehicle or positive) groups and in the test substance treated groups appeared normal during the study period.

 

• No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio) in the male and female test substance groups relative to the respective vehicle control groups were observed at 24 or 48 hours post-dose, suggesting that the test substance did not induce cytotoxicity.

 

• No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance 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, binomial distribution, Kastenbaum-Bowman Tables).

 

• CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs (p ≤ 0.05, binomial distribution, Kastenbaum-Bowman Tables) in both male and female mice.

 

• The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.

 

Under the conditions of the study conduct as described in this report, a single oral administration of the test substance at doses up to and including a dose of 2000 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female Crl:CD-1 (CD-1) mice. Therefore, the test substance was concluded to be negative in the mouse micronucleus assay.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mouse micronucleus assay
Species:
mouse
Strain:
other: Crl:CD1(ICR)
Details on species / strain selection:
Mice have been shown to exhibit micronuclei indicative of broken chromosomes (clastogenic effects) or spindle effects (aneugenic effects) in response to known mutagens and were therefore used in this assay. The Crl:CD1(ICR) mouse was selected based on extensive experience with this strain at DuPont Haskell and its suitability for genetic toxicology studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc. (Raleigh, North Carolina, U.S.A.)
- Age at study initiation: ~ 8 weeks old
- Weight at study initiation: Males: Solvent Control group: 31.2 g; Low-dose group: 31.1 g; Mid-dose group: 31.0 g; High-dose group: 31.6 g; and Positive control group: 31.0 g Females: Solvent Control group: 24.3 g; Low-dose group: 24.0 g; Mid-dose group: 24.2 g; High-dose group: 24.3 g; and Positive control group: 24.7 g
- Assigned to test groups randomly: Yes
- Fasting period before study: Not mentioned
- Housing: All animals were housed in solid-bottom cages with Enrich-o’Cobs™ (i.e., enrichment-containing bedding).
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): Tap water
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
0.1% Tween-80 in 0.5% aqueous methylcellulose
Duration of treatment / exposure:
All animals were given a single dose by oral gavage.
Frequency of treatment:
All animals were given a single dose by oral gavage.
Post exposure period:
No
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
The vehicle control and the low- and mid-dose groups contained 10 animals/sex. The high-dose group contained 14 animals/sex.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP)
Tissues and cell types examined:
Bone marrow smears were prepared immediately after the sacrifice
Evaluation criteria:
Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis. Further investigation of an equivocal result was not required to obtain a conclusive finding.

The test substance was judged negative if the following conditions were met:

• No statistically significant dose-related increases in the group mean MN-RETs above the concurrent vehicle control value occurred at any concentration of the test substance.
• The MN-RET values of the test substance-treated animals were within reasonable limits of the laboratory historical control range.

The test substance was judged positive if the following conditions were met:

• The group mean MN-RETs was statistically significantly increased at one or more concentrations of the test substance compared to the concurrent vehicle control values.
• An accompanying statistically significant dose-response increase in MN-RETs was observed.

Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. All micronucleus data analyses were one-tailed and conducted at a significance level of 5%. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.
Statistics:
Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. All micronucleus data analyses were one-tailed and conducted at a significance level of 5%. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.

For any treatment groups where the increase in MN-RETs was found to be statistically significant, the data were further analyzed for dose response using the Cochran-Armitage trend test.

For each treatment group, the mean and standard deviation of % RETs and % MN-RETs were calculated. Data were be transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation is appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the non-transformed proportion.

No statistical analysis was conducted on body weights or clinical signs.

See the section below for the Methods of Statistical Analyses used.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No clinical signs of toxicity were observed at any timepoint at any dose level in male or female animals exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No mortality occurred during the study.

No statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint (See Tables 1 and 2 in the following section for details).

A statistically significant depression in the frequency of PCE/total erythrocytes was observed in female mice at the 48 hour timepoint indicating target cell exposure. No other reductions in PCE frequency were detected at any other timepoint in either male or female animals exposed to the test substance.

The positive control groups exhibited a response consistent with the micronucleated PCE historical control data. There were no obvious changes in body weight or body weight gain in either male or female animals administered the test substance.

Table 1 - Micronucleus Evaluation for Male Mice

 

Parameter

Solvent Control

500 mg/kg Group

1000 mg/kg Group

2000 mg/kg Group

Positive Control Group

PCEs/1000 Erythrocytes

 

24-hour

 

48-hour

 

 

 

556 ± 14

 

565 ± 22

 

 

 

554 ± 43

 

a

 

 

 

563 ± 28

 

a

 

 

 

577 ± 16

 

562 ± 30

 

 

 

508* ± 17

 

b

PCE/NCE Ratio

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 1.255 ± 0.071

 

 

1.306 ± 0.112

 

 

 1.261 ± 0.229

 

 

a

 

 

 1.294 ± 0.149

 

 

a

 

 

 1.366 ± 0.092

 

 

1.289 ± 0.151

 

 

 1.032* ± 0.068

 

b

MNPCE/2000 PCEs

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 

3.0 ± 1.2

 

 

2.2 ± 1.9

 

 

 

2.0 ± 1.0

 

 

a

 

 

 

2.0 ± 1.0

 

 

a

 

 

 

1.8 ± 0.8

 

 

2.6 ± 1.9

 

 

 

35.3* ± 9.4

 

b

* Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test

a = Not evaluated at this timepoint

b = Group not included at this timepoint

 

Table 2 - Micronucleus Evaluation for Female Mice

 

PCEs/1000 Erythrocytes

 

24-hour

 

48-hour

 

 

 

566 ± 22

 

568 ± 11

 

 

 

568 ± 40

 

a

 

 

 

557 ± 16

 

a

 

 

 

557 ± 33

 

542* ± 12

 

 

 

516* ± 27

 

b

PCE/NCE Ratio

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 1.311 ± 0.127

 

 

1.317 ± 0.061

 

 

 1.333 ± 0.230

 

 

a

 

 

 1.258 ± 0.079

 

 

a

 

 

 1.265 ± 0.155

 

 

1.186* ± 0.059

 

 

 1.070* ± 0.118

 

b

MNPCE/2000 PCEs

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 

2.8 ± 1.5

 

 

1.8 ± 1.5

 

 

 

1.6 ± 0.9

 

 

a

 

 

 

1.8 ± 1.3

 

 

a

 

 

 

3.2 ± 2.6

 

 

1.8 ± 0.8

 

 

 

26.6** ± 7.6

 

 

b

 * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test

** Statistically significant difference from control at p < 0.05 by Dunn's test and the Dunnett/Tamhane-Dunnett test

a = Not evaluated at this timepoint

b = Group not included at this timepoint

Conclusions:
All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in animal bone marrow. The test substance was concluded to be negative in this in vivo study.
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) in male and female Crl:CD1(ICR) mice. Based on range-finding results, doses of 0, 500, 1000, and 2000 mg/kg of the test substance were selected for the main study. Concurrent control groups were administered 0.1% Tween-80 in 0.5% aqueous methylcellulose as the vehicle (negative) control, or 40 mg/kg of cyclophosphamide [positive control] (OECD Guidelines for the Testing of Chemicals No 474; EPA OPPTS Guideline 870.5395; EC Directive 2008/32/EC Method B.12; and MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3).

 

All animals were given a single dose by oral intubation. The vehicle control and the low- and intermediate-dose groups contained 10 animals/sex. The high-dose group contained 14 animals/sex. The positive indicator group consisted of 5 animals/sex. Half of the animals in each test substance and untreated control group were sacrificed at each timepoint, approximately 24 or 48 hours post-dosing, respectively. The positive control group was sacrificed approximately 24 hours post-dosing. Bone marrow smears were prepared immediately after the sacrifices. Two thousand PCEs per animal were evaluated for micronuclei and 1000 total erythrocytes per animal were evaluated for bone marrow toxicity.

 

Aliquots of the vehicle control and each test substance concentration were taken to confirm homogeneity, dose concentrations, and stability. Homogeneity and target concentrations were verified, and the test substance was stable for the duration of the dosing period.

 

No clinical signs of toxicity were observed at any timepoint at any dose level in male or female animals exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No mortality occurred during the study.

 

No statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint (See Tables 1 and 2 in the "Other information on results including tables" section for details).

A statistically significant depression in the frequency of PCE/total erythrocytes was observed in female mice at the 48 hour timepoint indicating target cell exposure. No other reductions in PCE frequency were detected at any other timepoint in either male or female animals exposed to the test substance.

 

The positive control groups exhibited a response consistent with the micronucleated PCE historical control data. There were no obvious changes in body weight or body weight gain in either male or female animals administered the test substance.

 

All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in animal bone marrow. The test substance was concluded to be negative in this in vivo study.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mouse micronucleus assay
Species:
mouse
Strain:
other: Crl:CD1(ICR)
Details on species / strain selection:
Mice have been shown to exhibit micronuclei indicative of broken chromosomes (clastogenic effects) or spindle effects (aneugenic effects) in response to known mutagens and were therefore used in this assay. The Crl:CD1(ICR) mouse was selected based on extensive experience with this strain at DuPont Haskell and its suitability for genetic toxicology studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc. (Raleigh, North Carolina, U.S.A.
- Age at study initiation: ~ 8 weeks old
- Weight at study initiation: Males: Control group: 32.2 g; Low-dose group: 31.3 g; Mid-dose group: 31.5 g; High-dose group: 30.9 g; and Positive control group: 31.1 g; Females: Control group: 23.6 g; Low-dose group: 24.4 g; Mid-dose group: 23.8 g; High-dose group: 23.7 g; and Positive control group: 23.9 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No data
- Housing: All animals were housed in solid-bottom cages with Enrich-o’Cobs™ (i.e., enrichment-containing bedding)
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): Tap water
- Acclimation period: 6 days for range-finding animals and 7 days for the definitive study animals

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
Route of administration:
oral: gavage
Vehicle:
0.1% Tween-80 in 0.5% aqueous methylcellulose
Details on exposure:
All animals were given a single dose by oral gavage. The vehicle control, low, intermediate, and positive control groups contained 5 animals/sex. The high-dose group contained 7 animals/sex.
Duration of treatment / exposure:
Single gavage dose
Frequency of treatment:
Single gavage dose
Post exposure period:
No
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
In the solvent control group, the positive control group, and 500 and 1000 mg/kg groups, there were 5 males and 5 females and in the 2000 mg/kg group, there were 7 males and 7 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP)
Tissues and cell types examined:
Peripheral blood samples were collected approximately 48 and 72 hours post-dosing for the test substance treated and vehicle control groups, and approximately 48 hours post-dosing for the positive control group. At least 20,000 RETs were analyzed per blood sample for the induction of micronuclei and toxicity as indicated by the frequency of immature erythrocytes (% RETs) among total erythrocytes (i.e., RETs plus normochromatic erythrocytes, NCEs).
Evaluation criteria:
Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis. Further investigation of an equivocal result was not required to obtain a conclusive finding.

The test substance was judged negative if the following conditions were met:

• No statistically significant dose-related increases in the group mean MN-RETs above the concurrent vehicle control value occurred at any concentration of the test substance.
• The MN-RET values of the test substance-treated animals were within reasonable limits of the laboratory historical control range.

The test substance was judged positive if the following conditions were met:

• The group mean MN-RETs was statistically significantly increased at one or more concentrations of the test substance compared to the concurrent vehicle control values.
• An accompanying statistically significant dose-response increase in MN-RETs was observed.

Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. All micronucleus data analyses were one-tailed and conducted at a significance level of 5%. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.
Statistics:
Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. All micronucleus data analyses were one-tailed and conducted at a significance level of 5%. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.

For Method of Statistical Analysis see the following section.

For any treatment groups where the increase in MN-RETs was found to be statistically significant, the data were further analyzed for dose response using the Cochran-Armitage trend test.

For each treatment group, the mean and standard deviation of % RETs and % MN-RETs were calculated. Data were be transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation is appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the non-transformed proportion.

No statistical analysis was conducted on body weights or clinical signs.

See the following section for Method of Statistical Analysis information.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No clinical signs of toxicity or mortality were observed in the range-finder experiment at 2000 mg/kg.

In the main study, no clinical signs of toxicity or test substance related mortality were observed at any dose level in male or female mice exposed to the test substances. No abnormalities were detected in the vehicle or positive control groups. There were no obvious changes in body weights or body weight gain in either male or female animals administered the test substance.

No statistically significant and biologically relevant increases in the micronucleated RET frequency were observed in any evaluated test substance treated group of male or female animals at either timepoint.

There were no statistically significant decreases in %RETs among the total erythrocytes observed in any evaluated test substance treated group of male or female animals at either timepoint.

The positive control group exhibited the expected responses resulting in statistically significant increases in micronucleated RETs that are consistent with the historical control data.

All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated RETs in mouse peripheral blood. The test substance was concluded to be negative in this in vivo study.

A statistically significant increase in MN-RETs was observed in female mice at the 48 hour timepoint; however, this increase was observed at the low dose only. A Cochran-Armitage trend test was conducted to evaluate the dose response trend and was found not to be significant. Therefore this increase was considered to be biologically irrelevant. No other statistically significant or biologically relevant increases in the MN-RET frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint.

Table 1 - Micronucleus Evaluation for Male Mice

 

 

Parameter

Solvent Control

500 mg/kg Group

1000 mg/kg Group

2000 mg/kg Group

Positive Control Group

RET (%)

 

48-Hour (Mean ± SD)

 

72-Hour (Mean ± SD)

 

 

2.06 ± 0.24

 

 

2.06 ± 0.22

 

 

2.18 ± 0.40

 

 

Not evaluated at this timepoint

 

 

2.41 ± 0.49

 

 

Not evaluated at this timepoint

 

 

2.99 ± 1.28

 

 

3.16 ± 1.25

 

 

0.55* ± 0.16

 

 Not included at this timepoint

MN-RET (%)

 

48-Hour (Mean ± SD)

 

72-Hour (Mean ± SD)

 

 

0.25 ± 0.07

 

 

0.21 ± 0.05

 

 

0.21 ± 0.06

 

 

Not evaluated at this timepoint

 

 

0.28 ± 0.16

 

 

Not evaluated at this timepoint

 

 

0.17 ± 0.03

 

 

0.19 ± 0.04

 

 

1.18** ± 0.32

 

Not included at this timepoint

 

* Statistically significant difference from control at p < 0.05 by Dunn's test.

** Statistically significant difference from control at p < 0.05 by Dunnett test.

 

Table 2 - Micronucleus Evaluation for Female Mice

 

 

Parameter

Solvent Control

500 mg/kg Group

1000 mg/kg Group

2000 mg/kg Group

Positive Control Group

RET (%)

 

48-Hour (Mean ± SD)

 

72-Hour (Mean ± SD)

 

1.48 ± 0.11

 

 

 

1.68 ± 0.42

 

2.07 ± 0.15

 

 

 

Not evaluated at this timepoint

 

1.60 ± 0.24

 

 

 

Not evaluated at this timepoint

 

1.39 ± 0.35

 

 

 

1.87 ± 0.24

 

0.63* ± 0.20

 

 

 Not included at this timepoint

MN-RET (%)

 

48-Hour (Mean ± SD)

 

72-Hour (Mean ± SD)

 

 

0.15 ±0.03

 

 

 

0.15 ± 0.07

 

 

0.26* ± 0.07

 

 

 Not evaluated at this timepoint

 

 

0.14 ± 0.01

 

 

 

Not evaluated at this timepoint

 

 

0.12 ± 0.05

 

 

 

0.16 ± 0.04

 

 

0.59* ± 0.19

 

 

 Not included at this timepoint

 

* Statistically significant difference from control at p < 0.05 by Dunnett test.

Conclusions:
The test substance was concluded to be negative in this in vivo study.
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow by analyzing peripheral blood reticulocytes (RETs) in male and female Crl:CD1(ICR) mice. Based on range-finding results, doses of 0, 500, 1000, and 2000 mg/kg of the test substance were selected for the main study. Concurrent control groups were administered 0.1% Tween-80 in 0.5% aqueous methylcellulose as the vehicle (negative) control, or 30 mg/kg of cyclophosphamide (positive control).

 

All animals were given a single dose by oral gavage. The vehicle control, low, intermediate, and positive control groups contained 5 animals/sex. The high-dose group contained 7 animals/sex. Peripheral blood samples were collected via tail vein bleeding, and a micronucleus evaluation was conducted by flow cytometry. Peripheral blood samples were collected approximately 48 and 72 hours post-dosing for the test substance treated and vehicle control groups, and approximately 48 hours post-dosing for the positive control group. At least 20,000 RETs were analyzed per blood sample for the induction of micronuclei and toxicity as indicated by the frequency of immature erythrocytes (% RETs) among total erythrocytes (i.e., RETs plus normochromatic erythrocytes, NCEs). All surviving animals were weighed and observed for mortality and clinical signs of toxicity at least daily.

 

Aliquots of the vehicle control and each test substance concentration were taken to confirm homogeneity, dose concentrations, and stability. All three test substance dose concentrations exceeded the targeted levels. Homogeneity was verified, and the test substance was determined to be stable for the duration of the dosing period.

No clinical signs of toxicity or mortality were observed in the rangefinder experiment at 2000 mg/kg.

 

In the main study, no clinical signs of toxicity or test substance related mortality were observed at any dose level in male or female mice exposed to the test substances. No abnormalities were detected in the vehicle or positive control groups. There were no obvious changes in body weights or body weight gain in either male or female animals administered the test substance.

 

No statistically significant and biologically relevant increases in the micronucleated RET frequency were observed in any evaluated test substance treated group of male or female animals at either timepoint.

 

There were no statistically significant decreases in %RETs among the total erythrocytes observed in any evaluated test substance treated group of male or female animals at either timepoint.

 

The positive control group exhibited the expected responses resulting in statistically significant increases in micronucleated RETs that are consistent with the historical control data.

 

All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated RETs in mouse peripheral blood. The test substance was concluded to be negative in this in vivo study.

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

Additional information

The test substance was evaluated in a battery of genotoxicity studies with four different test batches. Negative results were obtained in the bacterial gene mutation (Ames) assay and in the in vitro mammalian cell gene mutation test (CHO/HGRPT assay). In two early in vitro chromosome aberration studies, positive results were reported but with slightly conflicting results. In the first study, an increase in the percent of cells with chromosome aberrations occurred in the presence of metabolic activation. In a second study, an increase in abnormal cells was observed without metabolic activation. Clearly negative results were obtained in two subsequent in vitro chromosome aberration studies conducted with additional test batches which confirmed that the test substance is not clastogenic. Further, negative results were obtained in three separately conducted in vivo mouse bone marrow or peripheral blood micronucleus studies in male and female mice. The test batch which continued on to commercialization had negative findings in the Ames test, the chromosome aberration test and the in vivo micronucleus test. Therefore, the weight of evidence supports the conclusion that the test substance does not induce gene mutations or chromosome aberrations and can be concluded as not genotoxic.

Justification for classification or non-classification

Early studies with test batches which did not continue to commercialization showed some positive findings in the Ames and chromosome aberration studies. The batch used for commercialization had negative findings in the Ames test, the chromosome aberration test and the in vivo micronucleus test. With regards to all batches tested, the overall weight of evidence for the test substance was negative for mutagenicity and clastogenicity in vitro in bacterial and mammalian cells, respectively. Additionally, the test substance was negative when evaluated in vivo in laboratory animals. Based on an assessment of the robust genetic toxicity data for this substance, the substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.