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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study with acceptable restrictions. Lack of test material details and no historical control data.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
lack of test material details and no historical control data.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Physical state: colourless liquid
- Chemical denomination: glycerol monoacetate
- Analytical purity: assumed to be 100%
- Lot/batch No.: 38988
- Expiration date of the lot/batch: 1996-07-30
- Storage condition of test material: room temperature

Method

Target gene:
TK locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 buffered with sodium bicarbonate containing sodium pyruvate/Synperonic F68, L-glutamine and gentamicin
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:
Preliminary toxicity test
50, 150, 300, 625, 1250, 2500, 3750 and 5000 µg/mL
Experiment I/II
Without S9 mix: 300, 625, 1250, 2500, 3750 and 5000 µg/mL
With S9 mix: 700, 625, 1250, 2500, 3750 and 5000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: the miscibility of the test material in sterile deionized water was asses prior to the testing. It was confirmed that the test material was fully miscible with water at a concentration of 500 mg/mL.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methyl methanesulphonate, 10 µg/mL in water, without S9; 20-methylcholanthrene, 2.5 µg/mL in DMSO, with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 48 h
- Selection time: 11-12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-14 days

SELECTION AGENT: Selective medium consisted of cloning medium containing 4 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The criteria for a positive response were:
An increase in mutant frequency in treated cultures of at least 100 relative to the concurrent control. The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance. Evidence of a dose relationship over at least two dose levels, in any increase in mutant frequency. Demonstration of reproducibility in any increase in mutant frequency. The observed increases in mutant frequency must lie outside the historical control range with a corresponding RTG of 20%.
Statistics:
The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al (1989).

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: a decrease in pH from the control level of pH 7.5 to pH 6.0 at 5000 µg/mL was observed in both tests.

RANGE-FINDING/SCREENING STUDIES:
In the preliminary toxicity test, treatment with 50-5000 µg/mL in the absence and presence of S9 mix resulted in relative growth in suspension of 129-107% and 104-85% respectively compared to the solvent control.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Experiment I - in the absence of S9-mix.

Concentration

Relative Total Growth [%]

Growth in Suspension [%]

Mutants per 1E+06 surviving cells

[µg/mL]

0 (DMSO)

100

100

137

1250

86

86

128

2500

88

88

140

3750

88

86

118

5000

90

99

148

MMS, 10

46

84

657***

MMS = Methyl methane sulphonate

*** Significantly different from control P < 0.001

Table 2: Experiment II - in the absence of S9-mix. 

Concentration

Relative Total Growth [%]

Growth in Suspension [%]

Mutants per 1E+06 surviving cells

[µg/mL]

0 (DMSO)

100

100

174

1250

86

97

187

2500

104

99

179

3750

76

85

189

5000

92

97

176

MMS, 10

63

87

469***

MMS = Methyl methane sulphonate

*** Significantly different from control P < 0.001

Table 3: Experiment I - in the presence of S9-mix. 

Concentration

Relative Total Growth [%]

Growth in Suspension [%]

Mutants per 1E+06 surviving cells

[µg/mL]

0 (DMSO)

100

100

172

1250

100

97

133

2500

91

90

144

3750

83

78

169

5000

85

78

167

20-MC, 2.5

19

37

881***

20 -MC: 20 -Methylcholanthrene

*** Significantly different from control P < 0.001

Table 4: Experiment II - in the absence of S9-mix.

Concentration

Relative Total Growth [%]

Growth in Suspension [%]

Mutants per 1E+06 surviving cells

[µg/mL]

0 (DMSO)

100

100

128

1250

89

93

120

2500

79

82

130

3750

71

78

125

5000

62

79

131

20-MC, 2.5

16

34

775***

20 -MC: 20 -Methylcholanthrene

*** Significantly different from control P < 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative