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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 September 2008 - 04 January 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol tetrakis(3-mercaptopropionate)
EC Number:
231-472-8
EC Name:
Pentaerythritol tetrakis(3-mercaptopropionate)
Cas Number:
7575-23-7
Molecular formula:
C17H28O8S4
IUPAC Name:
3-[(3-sulfanylpropanoyl)oxy]-2,2-bis({[(3-sulfanylpropanoyl)oxy]methyl})propyl 3-sulfanylpropanoate
Details on test material:
- Name of test material (as cited in study report): Pentaerythritol tetra(3-mercaptoproprionate)
- Physical state: liquid
- Analytical purity: 96.6% (w/w)
- Purity test date: 2008-08-26
- Lot/batch No.: 24318
- Expiration date of the lot/batch: 2009-08-14
- Stability under test conditions: sensitive to oxidation
- Storage condition of test material: room temperature

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
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from male Wistar rats induced with phenobarbital (80 mg/kg bw) and ß-naphtoflavone (100 mg/kg bw) for three consecutive days by oral route.
Test concentrations with justification for top dose:
Experiment I
with metabolic activation: 40, 200, 300, 400, 450, 500, 550, 600 µg/mL
without metabolic activation: 10, 20, 30, 40, 50, 55, 60, 65 µg/mL

Experiment II
with metabolic activation: 130, 180, 230, 280, 480, 530, 630, 675 µg/mL
without metabolic activation: 20, 40, 50, 60, 80, 100, 110, 120 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulphonate 200 and 500 µg/mL, methylmethanesulfonate 10 µg/mL (without metabolic activation)
Positive control substance:
other: benzo(a)pyrene 3.5 µg/mL (with metabolic activation)
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4, 24 h
- Expression time (cells in growth medium): 72 or 48 h (5 h or 24 h exposure time)
- Selection time (if incubation with a selection agent): 6 days

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

NUMBER OF REPLICATIONS: 4

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- Other: colony sizing

OTHER:
Evaluation criteria:
There are several criteria for determining a positive result:
- clear and dose-reIated increase in the mutant frequency,
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher
than the historical range of negative controls) for at least one of the dose groups.
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low
Large/small colonies ratio (smaller than or equal to 1.5 times the ratio of clastogenic controls MMS and/or B[a]P) is an indication for potential
clastogenic effects rtnd/or chromosomal aberrations.
Statistics:
not necessary

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at >= 40 µg/mL (-S9); >=313 µg/mL (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

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

Concentration
[µg/ mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Mutation factor

Quotient large / small colonies

0 (DMSO)

100

100

60.71

1

1.25

40

99.45

98.54

44.95

0.74

200

99.45

71.69

43.25

0.71

300

96.70

62.66

68.92

1.14

400

91.76

54.59

107.48

1.77

450

98.90

48.87

64.57

1.06

500

89.01

32.29

112.17

1.85

0.87

550

95.60

27.59

85.69

1.41

1.26

600

90.11

13.21

108.15

1.78

1.10

B[a]P, 3.5

98.90

68.85

243.73

4.01

0.80

B[a]P  Benzo[a]pyrene

 

 

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration
[µg/ mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Mutation factor

Quotient large / small colonies

0 (DMSO)

100

100

46.33

1

2.23

10

81.87

75.76

89.19

1.93

20

97.25

82.59

54.28

1.17

30

95.05

69.19

43.11

0.93

40

65.60

57.56

68.01

1.47

50

86.81

38.86

78.51

1.69

55

100.00

34.93

57.96

1.25

1.31

60

95.60

25.04

48.16

1.04

2.19

65

96.70

16.34

79.48

1.72

1.47

EMS, 500

83.52

61.99

769.08

16.60

MMS, 10

93.41

68.16

332.63

7.18

0.84

EMS   Ethyl methane sulphonate

MMS  Methyl methane sulphonate

 

 

Table 3: Experiment II - 24 h Exposure - With Metabolic Activation

Concentration
[µg/ mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Mutation factor

Quotient large / small colonies

0 (DMSO)

100

100

95.25

1

5.45

130

111.60

93.47

65.40

0.69

180

111.60

86.53

46.30

0.49

230

107.84

77.71

71.18

0.75

280

96.55

66.27

93.14

0.98

480

114.11

40.44

49.36

0.52

530

110.97

30.48

67.17

0.71

1.39

630

105.96

18.22

79.49

0.83

2.04

675

89.03

13.89

164.89

1.73

2.58

B[a]P, 3.5

96.55

67.52

562.23

5.90

1.06

B[a]P  Benzo[a]pyrene

 

 

Table 4: Experiment II - 24 h exposure - Without Metabolic Activation

Concentration
[µg/ mL]

Cloning efficiency [%]

Relative Total Growth [%]

Mutants per 1E+06 cells

Mutation factor

Quotient large / small colonies

0 (DMSO)

100

100

58.93

1

3.13

20

92.39

95.48

109.56

1.86

40

92.39

89.64

102.58

1.74

50

99.15

77.81

72.05

1.22

60

95.77

57.17

79.05

1.34

80

93.52

34.35

108.22

1.84

100

98.03

29.14

72.35

1.23

3.93

110

103.10

22.42

82.57

1.40

3.00

120

96.90

10.59

63.38

1.08

2.15

EMS, 200

72.68

24.95

1539.16

26.12

MMS, 10

73.80

31.79

1207.82

20.50

0.90

EMS   Ethyl methane sulphonate

MMS  Methyl methane sulphonate

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without activation

PETMP is negative in the mouse lymphoma assay, with and without metabolic activation
Executive summary:

The test item PETMP was assessed for its potential to induce mutations at the thymidine kinase locus using the mouse lymphoma cellline L5178Y in accordance with OECD Guideline 476. The se1ection of the concentrations was based on data from the pre-experiment. In experiment I, 600 µg/mL (with metabolic activation) and 65 µg/mL (without metabolic activation) were selected as the highest

concentrations. In experiment II, 675 µg/ml. (with metabolic activation) and 120 µg/mL (without metabolic activation) were selected as the highest concentrations. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

with metabolic activation:

40, 200,300,400, 450, 500, 550, 600 µg/mL

and without metabolic activation:

10, 20, 30, 40, 50, 55, 60, 65 µg/mL

Experiment II

with metabolic activation:

130,180,230,280,480,530,630,675 µg/mL

and without metabolic activation:

20,40,50,60,80,100,110, 120 µg/mL

Growth inhibition was observed in experiment land II with and without metabolic activation.

In experiment I with metabolic activation the relative total growth (RTG) was 13.21% for the highest concentration (600 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 65 µg/mL with a RTG of 16.34%. In experiment II with metabolic activation the relative total growth (RTG) was 13.89% for the highest concentration (675 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 120 µg/mL with a RTG of 10.59%.

In experiment land II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

The positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item PETMP is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cellline L5178Y.

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