Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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-guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1991
Reference Type:
secondary source
Title:
Methanesulfinic acid, Aminoimino, CAS No: 1758-73-2
Author:
OECD SIDS
Year:
2002
Bibliographic source:
OECD SIDS; UNEP Publications

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted in 1984
GLP compliance:
yes (incl. certificate)
Remarks:
Central Veterinary Public Health Inspectorate (Section GLP), Rijswijk, The Netherlands
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
HGPRT-locus
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham´s F-12 culture medium, supplemented with heat-inactivated foetal calf serum (10% v/v), L-glutamine (2 mM) and gentamicin (50 mg/L) (growth medium)
- Properly maintained: yes- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: no
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Experiment I
Without and with S9 mix: 4.75, 13.7, 41.2,124, 370, 1111, 3333 and 10000 µg/mL (4 h)
Experiment II
Without S9 mix: 250, 500, 750, 1000, 1100, and 1200 µg/mL (4 h)
With S9 mix: 250, 500, 1000, 1250, 1500, 1750, 2000 and 2500 µg/mL (4h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ham´s F12-culture medium
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: dimethylnitrosamine and dimethylbenzanthracene
Remarks:
Ethylmethanesulfonate (EMS), 500 µg/mL culture medium, with S9; dimethylbenzanthracene (DMBA), 20 µg/mL, without S9 and dimethylnitrosamine (DMN) 3000 µg/mL
Details on test system and experimental conditions:
- Exposure duration: 4 h
- Expression time (cells in growth medium): about 24 h after addition of the test substance (day 1 of the study) cell cultures were trypsinized and counted. The initial survival of the cell was measured (Day 1 of the study). Furthermore, one 75 cm² tissue culture flask containing 1500000 (2 day culture period) or 750000 cells (3 day culture period) was prepared from the cell suspensions of each concentration level. The cells were subcultured at 2 or 3 day intervals during the expression period (till Day 8 of the study).
- Selection time (if incubation with a selection agent): Day 8
- Fixation time (start of exposure up to fixation or harvest of cells): Day 8
SELECTION AGENT: 6-TG
STAIN: 4% Giemsa
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The following criteria were used to evaluate the data obtained in the HGPRT assay:
a) the survival (absolute cloning efficiency) of negative controls should not be less than 50%<,
b) the mean mutant frequency of the negative control should fall within the range of 0 to 15 6-TG resistance mutants per 10E6 clonable cells
c) the mean mutant frequency of the positive controls should be higher than 20 6-TG resistant mutants per 10E6 clonable cells, and should at least be 5 times higher than the corresponding negative control
d) the highest test substance concentration should, if possible, result in a clear cytotoxic response (e.g: 10-30% of the relative initial survival). Any apparent increased in mutant frequency at concentrations of the test substance causing more than 90% toxicity is considered to be an artefact and not indicative of genotoxicity

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: - S9 at 1111.1 µg/mL; +S9: 3333.3 and 10000 µg/mL; Experiment II: at 1200 and 2500 µg/mL (+ and - S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese hamster Ovary (CHO)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration
[µg/mL]

Cloning efficiency [%]

Mutants frequency per 1E+06 clonable cells

0

104.4

1.9

4.75

100.5

0.5

13.7

99.2

0.0

41.2

101.7

1.5

124

92.1

0.0

370

99.5

1.0

1111

94.8

6.3

3333

--

--

10000

--

--

EMS 500

82.7

205.0

EMS  ethymethanesulfonate

Table 2: Experiment I - 4 h exposure - With Metabolic Activation 

Concentration
[µg/mL]

Cloning efficiency [%]

Mutants frequency per 1E+06 clonable cells

0

102.8

1.5

4.75

107.6

2.3

13.7

98.5

2.0

41.2

103.9

1.0

124

98.3

3.1

370

102.1

2.9

1111

94.4

2.6

3333

N.D (*)

--

10000

--

--

DMN 3000

74.5

93.3

DMHA 20

92.2

160.0

DMN  dimethylnitrosamine

DMBA dimethylbenzanthracene

N.D=not determined, because no cells were recovered after treatment of (*) because of a limited number of cells were recovered for cloning efficiency

Table 3: Experiment II - 4 h exposure - Without Metabolic Activation

Concentration
[µg/mL]

Cloning efficiency [%]

Mutants frequency per 1E+06 clonable cells

0

93.3

3.8

250

101.1

0.0

500

96.4

0.0

750

93.5

0.0

1000

93.6

0.0

1100

95.0

2.1

1200

97.2

1.0

EMS 500

78.9

67.2

EMS   ethymethanesulfonate

Table 4: Experiment II - 4 h exposure – With Metabolic Activation

Concentration
[µg/mL]

Cloning efficiency [%]

Mutants frequency per 1E+06 clonable cells

0

97.0

1.5

250

103.2

0.5

500

104.3

6.7

1000

96.0

3.1

1250

93.9

3.7

1500

95.4

1.6

1750

91.4

3.8

2000

91.2

3.3

2500

75.2

2.0

DMBA 20

85.5

164.9

DMBA dimethylbenzanthracene

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative