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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria (reverse mutation test (Ames), similar to OECD 471): negative with and without metabolic activation in S. typhimurium TA 1538, TA 1537, TA 1535, TA 100 and TA 98 and Saccharomyces cerevisiae D4

In vitro cytogenicity in mammalian cells (chromosome aberration, similar to OECD 473): negative with and without metabolic activation in Chinese hamster lung (CHL/IU) cells

In vitro gene mutation in mammalian cells (mouse lymphoma assay, OECD 476): negative with and without metabolic activation in mouse lymphoma L5178Y cells

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Strain to detect cross-linking mutagens missing, analytical purity of test substance not specified.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Strain to detect cross-linking mutagens missingy, analytical purity of test substance not specified.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
His-operon (S. typhimurium), ade/trp-loci (S. cerevisiae)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
Saccharomyces cerevisiae
Details on mammalian cell type (if applicable):
D4
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 fraction of Aroclor 1254-pretreated rats
Test concentrations with justification for top dose:
0.1, 1.0, 10.0, 100.0, 500.0 and 1000.0 µg/plate without activation; 0.1, 1.0, 10.0, 100.0 and 500.0 µg/plate with activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water, dimethylsulfoxid (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
TA1535, TA100, D4 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: QM, 10 µg/plate
Remarks:
TA1537 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA1538, TA98 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramin, 2.5 µg/plate
Remarks:
TA1535, TA1537, TA1538, TA98, TA 100 with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulfoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
D4 with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h for TA 1535, TA 1537, TA 1538, TA 98, TA 100; 3 - 5 days for D4

DETERMINATION OF CYTOTOXICITY
- Method: other: direct revertant colony counts
Evaluation criteria:
Strains TA 1535, TA 1537 and TA 1538: If the solvent control value is within the normal range, a chemical that produces a positive dose-response over three concentrations with the lowest increase equal to twice the solvent control value is considered to be mutagenic.
Strains TA 98, TA 100 and D4: If the solvent control value is within the normal range, a chemical that produces a positive dose response over three
concentrations with the highest increase equal to twice the solvent control value for TA 100 and 2 - 3 times the solvent control value for strains TA 98 and D4 is considered to be mutagenic. For these strains, the dose-response increase should start at approximately the solvent control value.

If a chemical produces a response in a single test that cannot be reproduced in one or more additional runs, the initial positive test data lose significance.
Statistics:
no data
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True 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:
cytotoxicity
Remarks:
Slightly toxic to TA 1537 at 500 µg per plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
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
True 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
True 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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Saccharomyces cerevisiae
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
The compound was slightly toxic to the strain TA 1537 at 500 µg per plate.
TA 98 was repeated at 100, 500 and 1000 µg/plate because of the increased number of revertants at 500 µg/plate over the background level. The repeat test was negative.
Conclusions:
Interpretation of results: negative
Executive summary:

The test substance did not demonstrate mutagenic activity in any of the assays conducted in this evaluation and was considered not mutagenic under these test conditions.

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:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagles Mimimal Essential Media with 10% Foetal Bovine Serum
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/β-Naphtoflavone-induced rat liver S9
Test concentrations with justification for top dose:
6(18)-h without S9: 4.89, 9.77, 19.53, 39.06, 78.13, 156.25 µg/mL
6(18)-h with S9: 4.89, 9.77, 19.53, 39.06, 78.13, 156.25 µg/mL
24-h without S9: 2.44, 4.89, 9.77, 19.53, 39.06, 78.13 µg/mL

Concentrations selected for metaphase analysis, highest concentration was lowest precipitating concentration:
6(18)-h without S9: 19.53, 39.06, 78.13 µg/mL
6(18)-h with S9: 19.53, 39.06, 78.13 µg/mL
24-h without S9: 9.77, 19.53, 39.06 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 and 24 h
- Expression time (cells in growth medium): 18 h after 6 h treatment

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 (100 per plate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
Cells with 38 or more chromosomes were classified as polyploid cells and the % incidence of polyploid cells reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. If there was a dose-related increase in endoreduplicated cells then they are reported separately. The percentage of cells showing structural chromosome aberrations (breaks and exchanges) was calculated and reported. The number of gap-type aberrations was recorded and reported.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary with the concurrent vehicle control value using Fisher‘s Exact test.
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A precipitate of the test material was seen at and above 78.13 µg/mL in both exposure groups and at and above 39.06 µg/mL in the 24 h continuous exposure group

RANGE-FINDING/SCREENING STUDIES: In all cases the test material showed no evidence of cell toxicity. The dose selection for the main experiments was based on the lowest precipitating dose level, which was 78.13 µg/mL for both short term exposure groups and 39.06 µg/mL for the continuous exposure group. An additional dose above the lowest precipitating dose was included for all exposures.

Table 1: Chromosome aberrations and cell viability

Test item

Concentration

Cell viability

Mean

Aberrant cells in %

 

in µg/mL

in %

mitotic index

Numerical

Structural

Exposure period 6 hrs without S9 mix

DMSO

--

100

100

0

0.5

MMC

0.1

78

97

0

52.0

EBS

19.53

93

149

0.5

2.0

39.06

87

104

0

1.0

78.13

90

110

0

0.5

Exposure period 24 hrs without S9 mix

DMSO

--

100

100

0

0

MMC

0.05

54

274

0

34.0

EBS

9.77

99

116

0

0.5

19.53

106

179

0

1.5

39.06

110

168

0

1.0

Exposure period 6 hrs with S9 mix

DMSO

--

100

100

0

0

CP

5.0

56

58

0

10.5

EBS

19.53

87

97

0

0

39.06

84

112

0

0

78.13

83

89

0

0

                       MMC: Mitomycin C

                       CP: Cyclophosphamide

                       EBS: Ethylene Bis(stearamide)

Conclusions:
Interpretation of results: negative
Executive summary:

The test material did not induce any statistically significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in the presence or absence of a liver enzyme metabolising system or after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 March - 27 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase (TK) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin, sodium pyruvate and L-glutamin + 10% (v/v) heat-inactivated horse serum (24-h exposure); for 3-h exposure only 5% (v/v) heat-inactivated horse serum were included. Selective medium consisted of the basic medium + 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphtoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1:
In the absence and presence of 8% (v/v) S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 3 h

Experiment 2:
In the absence of S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 24 h
In the presence of 12% (v/v) S9-mix: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 µg/mL for 3 h
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: hexane
- Justification for choice of solvent/vehicle: solubility/ability to suspend
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
hexane
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 h, respectively
- Expression time (cells in growth medium): 2 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT) (Sigma)
- Selection time (if incubation with a selection agent): 11 - 12 days

NUMBER OF REPLICATIONS: 5 exposure plates in 2 experiments

NUMBER OF CELLS EVALUATED: not applicable, number of mutants per well counted; 2000 cells/well inserted, total sum of mutants given

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth, relative survival, suspension growth, relative suspension growth, growth rate

Evaluation criteria:
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120%. An acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 x 10E-6 and ≤ 170 x 10E-6.
c) The growth rate (R) over the 2-day expression period for the negative controls should be between 8 and 32 (3 h treatment) and between 32 and 180 (24 h treatment).
d) The mutation frequency of MMS should not be below 500 x 10E-6, and for CP not below 700 x 10E-6.

The global evaluation factor (GEF) has been identified by the IWTG as the mean of the negative/solvent mutation frequeny (MF) distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than the MF of the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if: a) None of the tested concentrations reaches a mutation frequency of the MF of the controls + 126. b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility and precipitation: a slight precipitate of the test substance in the exposure medium was observed after 3 h treatment at the concentration of 0.8 µg/mL and severe precipitate was observed at concentrations of 2.4 µg/mL and above. The test substance was tested beyond the limit of the solubility to obtain adequate cytotoxicity data, the concentration used as the highest test substance concentration for the dose range Ending test was 24 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 24 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent treated control cultures were within the ranges of the historical controls.

Table 1: Cytotoxic and mutagenic responses

Treatment

Concentration [µg/mL]

Cloning efficiency [%]

Relative total growth [%]

Mutation frequency x 10-6

 

total

small colonies

large colonies

3 h treatment without S9-mix

Solvent control (mean)

--

99

100

114

62

46

Test substance

0

116

126

110

59

43

0.1

123

126

99

53

40

0.2

113

111

110

63

41

0.3

110

124

118

67

44

0.4

113

139

128

60

59

0.5

110

154

118

63

48

0.6*

108

125

126

71

48

0.8*

118

125

105

52

47

1.0*

115

125

119

67

45

MMS

15

58

55

1227

675

354

3 h treatment with 8% (v/v) S9-mix

Solvent control (mean)

--

106

100

102

55

42

Test substance

0

105

102

80

38

38

0.1

94

89

119

66

46

0.2

105

55

89

51

34

0.3

93

76

113

64

43

0.4

120

81

93

47

41

0.5

121

127

97

55

37

0.6*

101

86

87

53

30

0.8*

97

94

72

44

25

1.0*

121

112

79

45

30

CP

7.5

47

23

1431

876

351

24 h treatment without S9-mix

Solvent control (mean)

--

93

100

58

29

27

Test substance

0

86

138

66

34

31

0.1

72

108

77

43

32

0.2

79

121

78

38

38

0.3

98

150

47

26

19

0.4

90

151

55

27

26

0.5

81

133

69

37

30

0.6*

80

125

67

35

31

0.8*

90

143

61

37

22

1.0*

88

131

63

34

27

MMS

5

66

81

629

357

211

3 h treatment with 12% (v/v) S9-mix

Solvent control (mean)

--

106

100

82

50

29

Test substance

0

85

294

83

52

28

0.1

107

58

109

48

55

0.2

118

60

77

42

32

0.3

91

161

88

50

34

0.4

85

127

86

55

28

0.5

101

146

74

49

23

0.6*

86

42

103

66

32

0.8*

93

155

67

40

25

1.0*

62

66

130

55

70

CP

7.5

37

43

1905

1113

500

*precipitation of test substance in the exposure medium

Conclusions:
Interpretation of results: negative
Executive summary:

The test substance did not induce a significant increase in mutation frequency in the absence or presence of metabolic activation in the first experiment. This result was confirmed in an independent experiment with a different concentration of the metabolic activation system or a longer exposure time without activation. The test substance did not induce gene mutation in mammalian cells in vitro under the conditions of the study.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There is data from in vitro assays available addressing gene mutation in bacteria, clastogenic activity in mammalian cells and gene mutation in mammalian cells.

In vitro gene mutation in bacteria

Gene mutation in bacteria was assessed by the Reverse Mutation Assay (Ames test) conducted with 5 Salmonella typhimurium strains (TA1535, TA1537, TA1538, TA98 and TA 100) and the yeast strain Saccharomyces cerevisiae D4, which was comparable to OECD guideline 471 (Litton Biometics, 1978). All assays were conducted in presence and absence of a metabolic activation system consisting of Aroclor 1254-induced rat liver S9 with test substance concentrations ranging from 0.1 to 500 µg/plate; additionally 1000 µg/plate was included in a repetition test with TA98. Although there was no strain included to detect cross-linking mutagens according to the actual version of the guideline, the study was acceptable according to the standards used when the study was conducted. In this study no mutagenic effects on bacteria or yeast were observed with or without metabolic activation at any of the concentrations tested.

In vitro chromosome aberration

The clastogenic activity of the test substance was investigated by a Chromosomal Aberration Assay conducted with a Chinese Hamster lung cell line according to OECD guideline 473 (Safepharm, 2006). Duplicate cell cultures were exposed for either 6 h to concentrations up to 156.25 µg/mL in presence or absence of a metabolic activation system consisting of Phenobarbital/β-Naphtoflavone-induced rat liver S9-mix, followed by an 18-h recovery period, or for 24 h to concentrations up to 78.13 µg/mL in the absence of metabolic activation. Three concentrations and the vehicle control were chosen for analysis of 200 metaphases; the highest concentration chosen was the lowest one at which precipitation occurred. The chromosomes were analysed for structural aberrations comprising chromosome and chromatid breaks and exchanges, gaps, numerical aberrations and multiple aberrations. Under the conditions of this study the test substance did not induce any statistically significant, dose-related increases in the frequency of cells with structural or numerical chromosome aberrations either in presence or absence of a metabolic activation system after various exposure times. The test material was therefore considered to be non-clastogenic to CHL cells in vitro.

In vitro gene mutation in mammalian cells

Mutagenicity of the test substance in mammalian cells in vitro was determined in the L5178Y mouse lymphoma cell line according to OECD guideline 476 (NOTOX, 2010). The number of mutants derived from 5 exposure plates was determined in 2 independent exeriments. In the first experiment the mouse lymphoma cells were exposed for 3 h to test substance concentrations in the range of 0 to 1.0 µg/mL in the absence and presence of a metabolic activation system consisting of 8% (v/v) Phenobarbital/β-Naphtoflavone-induced rat liver S9-mix. In this setup precipitation occurred at concentrations ≥ 0.6 µg/mL, no cytotoxicity was observed, and there was no significant increase of mutant frequencies at the TK locus compared to the control groups. These findings were confirmed by a second independent experiment conducted with the same test substance concentrations, but with 3-h exposure in the presence of 12% (v/v) rat liver S9-mix or 24-h exposure without metabolic activation. In conclusion the test material did not induce gene mutations in mammalian cells in vitro.

According to Regulation (EC) No. 1907/2006, Annex IX, Item 8.4., Column 2, testing for genetic toxicity in vivo is not indicated as the test substance did not demonstrate any genotoxic activity in bacteria or mammalian cells in vitro.

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are therefore conclusive but not sufficient for classification.