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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:
experimental study
Adequacy of study:
key study
Study period:
03 March 2008 to 28 April 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP, with minor deviations; absence of clastogenic control (-S9) is not essential for the interpretation of the study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
a clastogenic positive control was not used -S9
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 2000
Deviations:
yes
Remarks:
a clastogenic positive control was not used -S9
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-mercaptopropionic acid
EC Number:
203-537-0
EC Name:
3-mercaptopropionic acid
Cas Number:
107-96-0
Molecular formula:
C3H6O2S
IUPAC Name:
3-sulfanylpropanoic acid
Details on test material:
- Name of test material (as cited in study report): 3-Mercaptopropionic acid
- Physical state: Clear colourless liquid
- Analytical purity: 99.8% (GC)
- Purity test date: 2008-02-21
- Lot/batch No.: 22333
- Expiration date of the lot/batch: 2009-02-19
- Stability under test conditions: no data
- Storage condition of test material: room temperature in the dark

Method

Target gene:
thymidine kinase
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from the livers of male Sprague-Dawley rats weighing ~250 g. These had each orally received three consecutive daily doses of phenobarbitone/ß-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on Day 4.
Test concentrations with justification for top dose:
Exp. 1 : +/- S9: 66.31, 132.63, 265.25, 530.5, 705.75, and 1061 µg/ml;
Exp 2:
4 hr w/ S9: 66.31, 132.63, 265.25, 530.5, 705.75, and 1061 µg/ml;
24 hr w/o S9: 16.5, 33, 66, 99, 132, and 198 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: serum-free culture medium
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9

Migrated to IUCLID6: 400 / 150 µg/mL (4 / 24 h exposure)
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: 2 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4, 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10-14 days

SELECTION AGENT (mutation assays): 4 µg/mL 5-trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.The IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set at 126E-06 for the microwell method. Therefore any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126E-06 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible
increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically
significant.
Statistics:
Linear trend analysis.

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
Remarks:
10 mM (1061 µg/mL)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
other: The positive controls caused an increase in mutation frequency, but no clastogenic positive control (e.g., MMS) was used in the absence of S9.
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the 4-hour exposure groups, both in the absence and presence of metabolic activation (S9), there was evidence of modest reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. However, a significant reduction in %RSG values of cells treated with test material was observed in the 24-hour exposure group in the absence of S9. No precipitate of the test material was observed at any of the dose levels. In the subsequent mutagenicity test the maximum dose level used was the 10 mM limit dose for the 4-hour exposure groups. However, for the 24-hour exposure group, the maximum dose level was limited by test material induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: controls were within historical reference range
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the
L5178Y cell line at the TK +/- locus.  The positive control materials induced marked increases in the mutant frequency
indicating the satisfactory performance of the test and the activity of the metabolizing system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency
at any dose level, either with or without metabolic activation, in either the first or the second experiment,
using a dose range that included the 10 mM dose in the 4-hour exposure groups and also a dose level that induced the optimum level of toxicity in the 24-hour exposure group.

The proportion of small colonies was not increased, indicating non-clastogenicity of the test material.

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

Concentration
[µg/ mL]

Relative Total Growth

Mutants per 1E+06 cells

Quotient small / large colonies

0

1.00

108.23

0.47

66.31

0.94

94.81

0.43

132.63

0.89

98.63

0.31

265.25

0.64

99.72

0.26

530.5

0.52

81.07

0.43

795.75

0.53

64.61

0.26

1061

0.49

90.63

0.40

EMS, 400

0.36

903.71

0.47

EMS:  Ethyl methane sulphonate

 

 

Table 2: Experiment 1- 4 h exposure - With Metabolic Activation

Concentration
[µg/ mL]

Relative Total Growth

Mutants per 1E+06 cells

Quotient small / large colonies

0

1.00

95.59

0.44

66.31

0.82

107.45

0.33

132.63

0.78

97.39

0.38

265.25

0.67

72.72

0.37

530.5

0.75

86.28

0.33

795.75

0.76

110.04

0.37

1061

0.80

112.26

0.39

CP, 2

0.23

941.38

0.77

CP: cyclophosphamide

 

 

Table 3: Experiment 2 - 24 h exposure - Without Metabolic Activation

Concentration
[µg/ mL]

Relative Total Growth

Mutants per 1E+06 cells

Quotient small / large colonies

0

1.00

102.67

0.28

16.5

1.54

65.04

0.31

33

1.26

91.52

0.21

66

1.15

76.53

0.22

99

0.94

78.61

0.27

132

0.34

105.28

0.14

198

0.11

89.77

0.15

EMS, 150

0.34

788.05

0.27

EMS: Ethyl methane sulphonate

 

 

Table 4: Experiment 2 - 24 h Exposure - With Metabolic Activation

Concentration
[µg/ mL]

Relative Total Growth

Mutants per 1E+06 cells

Quotient large / small colonies

0

1.00

95.61

0.21

66.31

0.94

94.73

0.29

132.63

0.70

100.86

0.21

265.25

0.57

84.05

0.17

530.5

0.54

95.55

0.25

795.75

0.49

71.98

0.27

1061

0.53

114.54

0.24

CP, 2

0.24

480.62

0.60

CP: cyclophosphamide

Applicant's summary and conclusion

Conclusions:
MPA was non-mutagenic to L5178Y cells under the conditions of the test. The proportion of small colonies was not increased, indicating non-clastogenicity of the test material.