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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
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
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
Target gene:
thymidine kinase
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
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.
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'.

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

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.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 March 2008 to 27 April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant, without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon (S.typhimurium)
trp operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
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:
50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without metabolic activation

Migrated to IUCLID6: 3 µg/plate for TA100, 5 µg/plate for TA1535 and 2 µg/plate for WP2uvrA-
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation

Migrated to IUCLID6: 80 µg/plate for TA1537
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation

Migrated to IUCLID6: 0.2 µg/plate for TA98
Positive control substance:
other: 2-Aminoanthracene, 1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate for WP2uvrA-
Remarks:
with metabolic activation
Positive control substance:
benzo(a)pyrene
Remarks:
with metabolic activation

Migrated to IUCLID6: 5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
Statistics:
n.a.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
Species / strain:
E. coli WP2 uvr A
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:

RANGE-FINDING/SCREENING STUDIES: The test material caused no visible reduction in the growth of the bacterial background lawn at any test concentration.

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

Table 1: Experiment 1 - Without Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

103

20

28

24

11

50

103

20

22

19

7

150

93

30

23

21

13

500

88

24

23

17

12

1500

84

25

17

18

10

5000

100

23

24

10

14

Pos controls
‑S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Conc. (µg/plate)

3

5

2

0.2

80

No. of revertants per plate

413

172

212

146

862

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

 

 

Table 2: Experiment 1 - With Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

83

11

18

29

12

+

50

75

15

28

27

13

+

150

83

13

28

22

9

+

500

92

11

25

20

8

+

1500

79

13

24

21

5

+

5000

70

0 V

25

15

5

Pos controls +S9

Name

2AA

2AA

2AA

BP

2AA

Conc. (µg/plate)

1

2

10

5

2

No. of revertants per plate

1481

211

496

187

249

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

V: Very weak bacterial background lawn

 

Table 3: Experiment 2 - Without Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

0

108

21

24

19

16

50

103

25

25

24

17

150

103

26

21

14

14

500

108

24

23

13

16

1500

107

26

25

18

16

5000

82

25

23

15

14

Pos controls
‑S9

Name

ENNG

ENNG

ENNG

4NQO

9AA

Conc. (µg/plate)

3

5

2

0.2

80

Avg. no. of revertants per plate

473

449

998

110

847

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

 

 

Table 4: Experiment 2 - With Metabolic Activation

S9 Mix

Test substance concentration (µg/ plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

TA102

TA98

TA1537

+

0

113

14

32

32

18

+

50

110

18

27

26

14

+

150

106

13

30

24

13

+

500

100

21

24

21

13

+

1500

80

16

25

19

10

+

5000

70

13

19

20

16

Pos controls +S9

Name

2AA

2AA

2AA

BP

2AA

Conc. (µg/plate)

1

2

10

5

2

No. of revertants per plate

1672

232

558

189

227

2AA: 2-Aminoanthracene

BP: Benzo(a)pyrene

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

MPA is negative in the Ames test, with and 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
Study period:
26 April 2007 to 17 July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP, no deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: not applicable, primary culture
- Periodically checked for Mycoplasma contamination: not applicable, primary culture
- Periodically checked for karyotype stability: not applicable, primary culture
- Periodically "cleansed" against high spontaneous background: not applicable, primary culture
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from the livers of male Sprague-Dawley rats weighing ~250 g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and ß-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
4-h exposure: 33.1 to 1060 µg/mL
24-h exposure: 33.1 to 530 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9

Migrated to IUCLID6: 0.4 and 0.2 µg/mL for 4- and 24-h exposure
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: 5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4, 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): 5% Gurrs Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear concentration-relationship. For modest increases in aberration frequency a concentration response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
Fisher's Exact test.
Species / strain:
lymphocytes: human
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The concentration range for the Preliminary Toxicity Test was 16.5 to 1060 µg/mL. The maximum concentration was the 10 mM concentration. No precipitate of the test material was observed at the end of the exposure period in any of the three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1060 µg/mL in the 4(20)-hour exposures both in the presence and absence of metabolic activation (S9). The maximum concentration with metaphases present in the 24-hour continuous exposure was 1060 µg/mL, though it should be noted that frequency of metaphases was very low. The test material induced some evidence of toxicity.


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

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.  All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.  The test material was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a concentration range that included a concentration level that induced approximately 50% mitotic inhibition.

MPA produced some evidence of toxicity at 1060 µg/mL in the preliminary  toxicity test, as well as in the chromosome aberration test.

Table 1: Experiment 1 - 4 h treatment, 20 h fixation - Without Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.0

0.5

0.0

265

91

0.0

1.5

0.5

530

75

0.5

0.0

0.0

1060

71

0.0

2.0

1.5

MMC, 0.4

60

0.0

35.3

33.3***

MMC: Mitomycin C

*** p<0.001

 

Table 2: Experiment 1- 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.0

1.0

0.5

265

100

0.0

0.5

0.5

530

69

0.0

0.5

0.5

1060

70

0.0

1.5

1.5

CP, 5

30

0.0

38.8

33.0***

CP: Cyclophosphamide

*** p<0.001

 

Table 3: Experiment 2- 20 h treatment, 20 h fixation - Without Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.5

0.5

0.5

66.25

104

0.0

2.0

0.5

132.5

82

0.0

2.5

0.5

265

36

0.0

3.0

2.0

MMC, 0.4

50

0.0

17.0

16.0***

MMC: Mitomycin C

*** p<0.001

 

 

Table 4: Experiment 2- 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.0

0.0

0.0

265

178

0.0

2.0

1.5

530

142

0.0

0.5

0.5

1060

161

0.0

2.5

0.5

CP, 5

54

0.0

28.0

24.0***

CP: Cyclophosphamide

*** p<0.001

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

The test material is non-clastogenic to human lymphocytes in vitro, with and without metabolic activation.

Additional information

MPA was negative, with and without metabolic activation in an Ames test, a mouse lymphoma assay, and a chromosomal aberration assay in human lymphocytes in vitro.

Short description of key information:

3-Mercaptopropionic acid (MPA) was negative in a complete battery of in-vitro genotoxicity assays.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification