Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

PETMA is considered non-genotoxic in vitro based on read-across from structurally related compounds.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached justifications
Reason / purpose:
read-across source
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
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:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9, induced with phenobarbitone/ß-naphthoflavone (80/100 mg per kg per day)
Test concentrations with justification for top dose:
50 to 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with poor water solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without activation Migrated to IUCLID6: 3 µg/plate for TA100 and 5 µg/plate for TA1535
Positive control substance:
9-aminoacridine
Remarks:
without activation Migrated to IUCLID6: 80 µg/plate for TA1537
Positive control substance:
mitomycin C
Remarks:
without activation Migrated to IUCLID6: 0.5 µg/plate for TA102
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without 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
Remarks:
with activation
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 5 µg/plate for TA98
Positive control substance:
other: 1,8-Dihydroxyanthraquinone, 10 µg/plate for TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: n.a.
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 per concentration per experiment
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.
Statistics:
Dunnett's method of linear regression
Key result
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000 µg/plate
- Other confounding effects:


RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100).

COMPARISON WITH HISTORICAL CONTROL DATA:
pos. and neg. controls within historical 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

TA102

TA98

TA1537

0

82

25

383

18

12

50

77

22

352

15

10

150

75

22

359

17

7

500

80

21

365

16

9

1500

71

21

346

18

9

5000

74 P

33 P

325 P

18 P

7 P

Pos controls
‑S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Conc. (µg/plate)

3

5

0.5

0.2

80

No. of revertants per plate

270

444

1186

171

1287

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

MMC  Mitomycin C

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

TA102

TA98

TA1537

+

0

87

13

375

46

19

+

50

84

9

365

37

13

+

150

80

10

356

35

18

+

500

77

13

345

30

14

+

1500

62

14

319

24

13

+

5000

51 P

16 P

233 P

32 P

8 P

Pos controls +S9

Name

2AA

2AA

DAN

BP

2AA

Conc. (µg/plate)

1

2

10

5

2

No. of revertants per plate

1853

351

784

225

329

2AA    2-Aminoanthracene

BP     Benzo(a)pyrene

DAN   1,8-Dihydroxyanthraquinone

P        Precipitate

 

 

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

TA102

TA98

TA1537

0

76

22

353

15

10

50

81

23

368

14

7

150

69

29

373

14

8

500

78

30

351

18

9

1500

90

30

343

23

8

5000

94 P

36 P

280 P

24 P

4 P

Pos controls
‑S9

Name

ENNG

ENNG

MMC

4NQO

9AA

Conc. (µg/plate)

3

5

0.5

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

MMC  Mitomycin C

P        Precipitate

 

 

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

87

15

376

30

12

+

50

75

15

395

23

15

+

150

76

17

384

22

11

+

500

69

17

382

22

9

+

1500

60

16

338

19

6

+

5000

54 P

12 P

157 P

21 P

9 P

Pos controls +S9

Name

2AA

2AA

DAN

BP

2AA

Conc. (µg/plate)

1

2

10

5

2

No. of revertants per plate

2950

168

748

152

264

2AA    2-Aminoanthracene

BP     Benzo(a)pyrene

DAN   1,8-Dihydroxyanthraquinone

P        Precipitate

 

 

 

Conclusions:
Based on read-across from PETMP, PETMA is considered negative in the Ames test, with and without metabolic activation
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached justifications
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
August 1998
Deviations:
not specified
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
- 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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 Migrated to IUCLID6: 200 and 500 µg/mL
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 10 µg/mL
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 Migrated to IUCLID6: 3.5 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): 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
Key result
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

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

 

Conclusions:
Based on read across from PETMP, PETMA is predicted to be negative in the mouse lymphoma assay, with and without metabolic activation
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached justifications
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
August 1998
Deviations:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
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: 62.5, 125 and 190 µg/mL
without metabolic activation: 1.95, 3.9 and 7.8 µg/mL
Experiment II:
with metabolic activation: 200, 275 and 300 µg/mL
without metabolic activation: 0.49, 0.98 and 1.95 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without activation Migrated to IUCLID6: 400 and 600 µg/mL
Positive control substance:
cyclophosphamide
Remarks:
with activation Migrated to IUCLID6: 0.83 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: n.a.
- Exposure duration: 4, 20 h
- Expression time (cells in growth medium): n.a.
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid(R)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell density

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the concentrations, which is higher than the laboratory negative control range (0.0% - 4.5%
aberrant cells (with metabolic activation) and 0.0% - 4.0% aberrant cells (without metabolic activation)).
Statistics:
not applicable
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 7.8/190 µg/mL, -/+ S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 250 µg/mL and higher.

RANGE-FINDING/SCREENING STUDIES:
According to the used guidelines the highest recommended concentration is 5000 µg/mL. The test item was dissolved in DMSO and diluted in cell
culture medium. The highest concentration evaluated in the preexperiment was 5000 µg/mL. The relative mitotic index was used as parameter for toxicity. The concentrations evaluated in the main experiment based on the results obtained in the pre-experiment-

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

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

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

0 (DMSO)

100

2

6

3

1.95

89

4

8

5

3.9

89

6

10

4

7.8

20

0

9

4

EMS, 600

114

2

27

17

EMS: Ethyl methane sulphonate

 

 

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

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

0 (DMSO)

100

0

6

4

62.5

94

2

9

5

125

95

1

7

2

190

30

2

14

5

CPA, 0.83

79

2

35

24

CPA: Cyclophosphamide

 

 

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

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

0 (DMSO)

100

0

13

7

0.49

105

2

8

3

0.98

84

2

10

3

1.95

32

3

10

4

EMS, 400

102

2

24

20

EMS: Ethyl methane sulphonate

 

 

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

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells

Aberrant cells

incl. gaps

excl. gaps

0 (DMSO)

100

2

3

1

200

73

6

14

7

275

39

5

12

4

300

33

5

6

1

CPA, 0.83

90

2

23

18

CPA: Cyclophosphamide

Conclusions:
Based on read-across from PETMP, PETMA does not induce chromosomal aberrations in mammalian cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

PETMA is considered non-genotoxic in vivo based on read-across from PETMP.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Justification for type of information:
see category report attached as "full study report"
Key result
Sex:
not specified
Genotoxicity:
negative
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: read across
Conclusions:
negative based on read across
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

Read-across from PETMP does not suggest a genotoxic hazard for PETMA.