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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: 2000/32/EG, B.17 (HPRT Test); OECD 476 (1997)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation (B17)
Species / strain / cell type:
mammalian cell line, other: Chin. Hamster, Ovarienzellen
Remarks:
mammalian cell line
Metabolic activation system:
Aroclor-induced rat liver S-9 mix.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 6.3 ... 5000 µg/ml
Concentration range in the main test (without metabolic activation): 3.1 ... 100 µg/ml
Vehicle / solvent:
DMSO 1 % (v/v)
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 4 hours
Expression time:
After an entire expression period of 6 - 8 days the cells
were transferred into selection medium (2nd passage).
Selection time:
6 - 8 days.
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(> 100 µg/ml)
Additional information on results:
Observations:
Precipitations with and without S-9 mix from 50 µg/ml
upwards.
Remarks on result:
other: main test Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: Siehe Bemerkung
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cytogenicity (B10)
Species / strain / cell type:
mammalian cell line, other: Chin. Hamster, Ovarienzellen
Remarks:
mammalian cell line
Metabolic activation system:
without
Test concentrations with justification for top dose:
Concentration range in the main test (without metabolic activation): 32.77 ... 80 µg/ml
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
Exposure period (without metabolic activation): 2 hours
Species / strain:
mammalian cell line, other: Chin. Hamster, Ovarienzellen
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Additional information on results:
Observations:
Keine toxischen Effekte.
Ohne S9-Mix: Ab 51,2 µg/ml Präzipitationen
«ENGLISCH+
No findings
Without S9-mix: Up to 51,2 µg/ml precipitations
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Experimental Toxicology and Ecology BASF AG
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 medium including 10% (v/v) FCS
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:

Aroclor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
1st Experiment (4h-exposure)
- without S9 mix: 0; 6.3; 12.5; 25.0; 50.0; 100.0 µg/mL
- with S9 mix: 0; 12.5; 25.0; 50.0; 2500.0; 3750.0; 5000.0 µg/mL
2nd experiment:
- without S9 mix (24-h exposure period): 0; 3.1; 6.3; 12.5;25.0; 50.0; 75.0; 100.0 µg/mL
- with S9 mix (4-h exposure period): 0; 6.3; 12.5; 25.0; 50.0; 100.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance on water, DMSO was selected as the vehicle, whcih had been demonstrated to be suitable in the CHO/HPRT test and for which historical data are available
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 10 µg/mL methylchlolanthrene dissolved in DMSO
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4, 24 h
- Expression time (cells in growth medium): about 3 days (4-h treatment) or 2 days (24-h treatment). This was followed by the 1st passge. After an entire expression period of 6 - 8 days the cells were transferred into selection medium (2nd passage)
- Selection time (if incubation with a selection agent): about 6 - 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): 6-thioguanine (10 µg/mL)

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 200

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
The HPRT assay was considered valid if the following criteria were met:
- The absolute cloning efficiencies of the negative controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative controls should fall within our historical negative control data range of 0 – 15 mutants per 10^6 clonable cells.
- The positive controls both with and without S9 mix had to induce distinctly increased mutant frequencies.
- At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances were not tested at concentrations higher than 5 mg/mL or 10 mM.

A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies both above the concurrent negative control values and our historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose response relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity and the molecular weight of the test substance 5 000 μg/mL (approx. 6.08 mM) was used as top concentration both with and without S9 mix at 4-hour exposure time and without S9 mix at 24-hour exposure time. After 4 hours treatment in the absence of S9 mix no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed. In contrary, in the presence of S9 mix, strongly reduced relative cloning efficiency of below 20% relative survival was observed after treatment with 5 000 μg/mL. In addition, after 24 hours treatment in the absence of S9 mix strongly reduced relative cloning efficiency of below 20% relative survival was observed after treatment with 100 to 2 500 μg/mL but not at the top dose, i.e. 5000 µg/ml.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results:

Experiment

Exposure period

Concentration (µg/ml)

S9 mix

Precipitation

Genotoxicity MFcorr /106cells

Cytotoxicity

CE1

CE2

1

4 h

vehicle

-

-

3.78

100.0

100.0

6.3

-

-

1.79

120.5

100.1

12.5

-

-

1.84

110.0

92.1

25

-

-

0.90

105.2

94.8

50

-

+

2.97

100.9

89.1

100

-

+

2.15

100.7

92.6

EMS

-

-

51.57

103.7

82.7

 

 

 

 

 

 

 

4 h

vehicle

+

-

3.83

100.0

100.0

12.5

+

-

3.64

80.8

96.1

25

+

-

0.63

78.7

102.9

50

+

+

2.08

72.3

107.9

2500

+

+

1.09

71.5

108.8

3750

+

+

0.31

102.5

109.1

5000

+

+

2.09

107.6

106.3

MCA

+

-

60.43

127.5

99.4

 

 

 

 

 

 

 

 

2

24 h

untreated control

-

-

2.88

98.8

109.3

vehicle

-

-

4.19

100.0

100.0

3.1

-

-

0.42

96.4

96.6

6.3

-

-

3.29

93.0

107.2

12.5

-

-

0.98

93.8

122.6

25

-

-

0.76

80.6

104.7

50

-

+

0.00

46.8

93.6

75

-

+

0.26

27.5

128.1

100

-

+

0.00

9.5

110.5

EMS

-

-

309.69

60.9

93.1

 

 

 

 

 

 

 

4 h

vehicle

+

-

1.09

100.0

100.0

6.3

+

-

1.50

92.1

83.1

12.5

+

-

0.98

96.2

91.1

25

+

-

1.32

96.9

87.8

50

+

+

6.03

86.0

85.6

100

+

+

1.04

74.5

84.4

MCA

+

-

116.16

81.5

87.2

CE: cloning efficiency = total number of colonies in the test group / total number of seeded cells in the test group x 100

CE was related to respective negative control (%)

MFcorr.: number of mutant colonies per 10^6 cells corrected with the CE2 value

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: 92/69/EWG, B.10 (Säuger zytogenetischer in vitro-Test); OECD 473
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cytogenicity (B10)
Species / strain / cell type:
mammalian cell line, other: Chin. Hamster, V79-Zellen
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 10 ... 100 µg/ml
Concentration range in the main test (without metabolic activation): 10 ... 100 µg/ml
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 18 hours
Expression time:
«ENGLISCH+
Fixation time:
Mit und ohne S9-Mix: 18 und 28 Stunden
«ENGLISCH+
With and without S9-mix: 18 and 28 h
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Additional information on results:
Observations:
Mit und ohne S9-Mix: Keine toxischen Effekt
Mit und ohne S9-Mix: Präzipitationen ab 33 µg/ml
«ENGLISCH+
With and without S9-mix: No findings
With and without S9-mix: precipitation up to
33 µg/ml
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: Siehe Bemerkung
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
bacteria, other: esc WP2
Metabolic activation system:
none
Test concentrations with justification for top dose:
Concentration range in the main test (without metabolic activation): 1.6 ... 1000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 1000 µg/plate
Species / strain:
other: esc WP2
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Additional information on results:
Observations:
o.B.
«ENGLISCH+
No findings
Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: OECD 471
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
bacteria, other: sat TA 98, 100, 1535, 1537
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 20 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 20 ... 5000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Species / strain:
other: sat TA 98, 100, 1535, 1537
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification