Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from category member HMDTMP-xNa: negative with and without activation in Salmonella typhimurium strains TA98 and TA100 (similar to OECD Test Guideline 471) (Department of Biochemistry, University of Birmingham, 1981).

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from category member HMDTMP-H: negative with and without activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 (similar to OECD Test Guideline 471) (Monsanto, 1976).

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from analogue substance DTPMP-H: negative in Salmonella typhimurium strains TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (similar to OECD Test Guideline 471) (BMI Inc, 2003).

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from analogue substance DTPMP-7Na: negative in Salmonella typhimurium strains TA98, 100, 1537 and 1535 and in Escherichia coli strain WP2 uvrA (similar to OECD Test Guideline 471) (Japan Oilstuff Inspectors Corporation, 2001).

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from EDTMP-5Na: negative with and without activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 (similar to OECD Test Guideline 471) (Monsanto, 1981a).

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from EDTMP-Ca-Na salt: negative with and without activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 (similar to OECD Test Guideline 471) (Monsanto, 1981b).

Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from EDTMP-xNa: negative with and without activation in Salmonella typhimurium strain TA 102 (OECD Test Guideline 471) (Harlan Cytotest Cell Research, 2012).

Cytogenicity in mammalian cells: read-across from closely-related substance EDTMP-H: negative with and without metabolic activation in CHO cells (similar to OECD Test Guideline 473) (Monsanto, 1986a).

Cytogenicity in mammalian cells: read-across from DTPMP-7Na; positive in Chinese hamster lung IU cells (OECD Test Guideline 473) (Japan Oilstuff Inspectors Corporation, 2001)

Mutagenicity in mammalian cells: HMDTMP (4-7K) was negative with and without metabolic activation in L5178Y mouse lymphoma cells (similar to OECD Test Guideline 476) (Litton Bionetics, 1978)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The restrictions were that only a short exposure time was used. It was not compliant with GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
only short exposure time used
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
5-30 µl/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given in study report
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation 0.5 µl/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine 0.3 µl/ml
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): selection medium containing 5% v/v BrdU

NUMBER OF REPLICATIONS: triplicate plates

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

ACTIVATION: NADP and isocitric acid were used as co-factors. Final concentration of S9 is not clearly indicated in the report.
Evaluation criteria:
A substance is considered mutagenic if there is a dose-related increase over 3 concentrations with the minimum increase at least 2.5 times the solvent and/or negative control values.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
20 µl/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Results of mutagenicity assay (mean of three plates)

Concentration µl/ml

Relative cloning efficiency

Relative total growth

Mutant frequency

-MA

+MA

-MA

+MA

-MA

+MA

Negative control

100

100

100

100

9.0

7.0

Solvent control

92.2

102.4

67.0

102.8

15.5

14.3

5

68.4

71.7

74.7

81.2

8.8

8.6

10

79.9

100.8

62.2

80.8

6.4

14.1

15

73.6

82.5

64.4

83.5

15.6

7.3

20

60.0

40.7

57.1

36.7

13.6

4.3

30

5.4

2.0

3.9

2.2

28.3

9.6

Positive control

35.4

7.3

18.0

0.3

180.4

700

Conclusions:
HMDTMP (4-7K) has been tested according to a protocol that is similar to OECD 476. No increase in the mutant frequency was observed at any concentration up to cytotoxic concentrations, with and without metabolic activation. Appropriate positive, solvent and negative controls were included and gave the expected results. It is concluded that the substance is negative for mutagenicity to L5178Y mouse lymphoma cells under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15-Jun-2001 to 18-Sep-2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines on Industrial Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
mammalian cell line, other: CHL/IU (originally derived from female Chinese hamster lung)
Details on mammalian cell type (if applicable):
- Type and identity of media: Eagle's minimum essential medium
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital- and 5,6-benzoflavone-induced male rat liver S9
Test concentrations with justification for top dose:
625-5000 µg mixed sodium salts/ml (pulse treatment)
150-3000 µg mixed sodium salts/ml (24-hour continuous treatment)
50-400 µg mixed sodium salts/ml (48-hour continuous treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: saline
- Justification for choice of solvent/vehicle: solubility of test substance in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9, 0.1 µg/ml (pulse treatment), 0.03 µg/ml (continuous treatment)
Positive control substance:
mitomycin C
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9, 15 µg/ml (pulse treatment)
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: pulse treatment 6 hours; continuous treatment, 24 or 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): pulse treatment 24 hours; continuous treatment 24 or 48 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.1 µg/ml, 2 hours
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 100 cells /culture, 2 cultures/treatment

DETERMINATION OF CYTOTOXICITY
- Method: other: cell density after crystal violet staining

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: mitotic index
Evaluation criteria:
Negative: both the incidence of aberrant cells (excluding gaps) and that of numerical aberrations <5%
Inconclusive: either of these incidences is 5-10%
Positive: either of these incidences >10%
Statistics:
None
Key result
Species / strain:
mammalian cell line, other: CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
pulse treatment ( 6 and 24 hour exposure)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=2500 µg mixed sodium salts/ml (pulse treatment),
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
mammalian cell line, other: CHL/IU
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
48 hour treatment
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=50 µg mixed sodium salts/ml (48-hour continuous treatment)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- No, but cell survival was evaluated at 156-5000 µg mixed sodium salts/ml (pulse treatment, 24-hour continuous treatment and 48-hour continuous treatment) and the data used to determine the concentrations that were to be evaluated for chromosome aberrations

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cell survival:
- Pulse treatment -S9, µg mixed sodium salts/ml: 156 (109%), 313 (109%), 625 (97%), 1250 (96%), 2500 (76%), 5000 (58%)
- Pulse treatment +S9, µg mixed sodium salts/ml: 156 (98%), 313 (104%), 625 (103%), 1250 (108%), 2500 (102%), 5000 (77%)
- 24-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (115%), 313 (81%), 625 (47%), 1250 (37%), 2500 (19%), 5000 (0%)
- 48-hour continuous treatment -S9, µg mixed sodium salts/ml: 156 (50%), 313 (25%), 625 (7%), 1250 (5%), 2500 (1%), 5000 (0%)

Full details of the results from the 48 hours treatment (result reported as positive) are not presented in the study report. A graph of the results indicates that the incidence of cells with structural aberrations was 2.5% at 50 µg/ml; 5% at 100; 15% at 150; 37% at 200 and 49% at 300 µg/ml.

Table 1 Chromosome aberration test - treatment time 6 hours

Concentration µg/ml

+/- S9 mix

No. of cells analyzed

Chromatid breaks

Chromatid exchanges

No. of cells with aberrations (%)

No. of cells with gaps

Cell growth index (%)

Polyploid cells

Control

-

200

0

0

0

1

100

0

625

-

200

1

0

1

0

98

0

1250

-

200

3

1

4

0

91

0

2500

-

200

6

1

7

4

71

2

5000

-

200

9

1

9

3

41

0

Positive control

-

200

54

121

139

1

-

0

Control

+

200

0

0

1

0

100

0

625

+

200

0

2

2

1

91

0

1250

+

200

0

1

1

0

95

0

2500

+

200

3

3

4

0

99

0

5000

+

200

1

0

1

0

67

0

Positive control

+

200

7

42

46

1

-

0

 

Chromosome aberration test - treatment time 24 hours

Concentration µg/ml

+/- S9 mix

No. of cells analyzed

Chromatid breaks

Chromatid exchanges

No. of cells with aberrations (%)

No. of cells with gaps

Cell growth index (%)

Polyploid cells

Control

-

200

2

0

2

1

100

0

4.7

-

200

1

1

2

1

101

0

9.4

-

200

2

0

2

0

102

0

18.8

-

200

4

0

4

1

104

0

37.5

-

200

3

2

5

0

107

0

75

-

200

2

1

3

3

103

0

150

-

200

8

0

8

2

113

0

300

-

-

TOXIC

Positive control

-

200

30

30

58

0

-

0

Control

+

200

2

0

2

0

100

0

50

+

200

4

1

5

0

99

0

100

+

200

7

4

11

2

68

0

150

+

200

31

9

34

0

57

0

200

+

200

67

9

74

1

43

0

300

+

200

87

10

97

2

20

0

400

+

-

TOXIC

Positive control

+

200

47

56

86

0

-

0

 

Conclusions:
DTPMP-7Na, has been tested for clastogenicity in a reliable study, conducted according to OECD Test Guideline 473 and in compliance with GLP. A dose related increase in the number of cells with aberrations was observed aftter 48 hours treatment in an in vitro chromosome aberration assay. The substance was concluded to be positive for the induction of chromosome aberrations. Appropriate solvent and positive controls were included and gave expected results.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02-Jul-2001 to 26-Jul-2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 2-AA used as positive control +S9)
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines on Industrial Chemicals
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay
Target gene:
histidine (Salmonella typhimurium)
tryptophan (Escherichia coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital- and 5,6-benzoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Test 1-1: 78-5000 µg mixed sodium salts/plate -S9, 156-5000 µg/plate +S9
Test 1-2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium
Test 2: 20-5000 µg mixed sodium salts/plate -S9 for S typhimurium, 78-5000 µg mixed sodium salts/plate -S9 for E coli, 156-5000 µg mixed sodium salts/plate +S9
20-5000ug/plate -S9; 156-5000ug/plate +S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9; TA98, 0.1 µg/plate; TA100, 0.01 µg/plate
Positive control substance:
furylfuramide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9 ; TA1535, 0.5 µg/plate
Positive control substance:
sodium azide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9; TA1537, 80 µg/plate
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
without S9, WP2 uvrA, 2 µg/plate
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9; TA98, 0.5 µg/plate; TA100, 1 µg/plate; TA1535, TA1537, 2 µg/plate; WP2 uvrA, 10 µg/plate
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition
Evaluation criteria:
Positive: mean number of revertant colonies more than double the negative control value and signlflcant concentration-dependent Increase
Statistics:
No statistical analysis performed
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:
cytotoxicity
Remarks:
>= 625 µg mixed sodium salts/plate -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=1250 µg mixed sodium salts/plate -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no precipitation observed
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations of 5-5000 µg/plate
- No growth inhibition in any strain
- Bacteriostatics observed at 1250 µg/plate -S9 in all strains
- No precipitation

COMPARISON WITH HISTORICAL CONTROL DATA:
- Negative/solvent control: within the range of historical data
- Positive controls: within the range of historical data

All treated cultures had less than 2-fold increase in mutant frequency and no evidence of a dose-response.  All positive controls gave a greater than 2-fold increase in mutant numbers.

Table 1 Experiment 1 Mean number of revertants (average of 3 plates)

 

Concentration (µg/plate)

+/- S9 mix

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

Control

-

102

10

23

14

7

78

-

102

8

21

16

9

156

-

88

7

18

16

6

313

-

71

5

18

8

1

625

-

0

0

7

0

0

1250

-

0

0

0

0

0

2500

-

0

0

0

0

0

5000

-

0

0

0

0

0

Positive control

-

528

422

631

559

194

Control

+

111

11

25

24

6

156

+

133

11

30

24

10

313

+

148

11

21

22

6

625

+

140

9

23

17

6

1250

+

122

6

21

11

3

2500

+

108

4

15

9

3

5000

+

98

5

9

8

2

Positive control

+

501

260

648

446

154

Mean number or revertants 16-19.07.2001

Table 2 Experiment 2 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)

+/- S9 mix

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

Control

-

104

11

-

16

7

20

-

101

6

-

16

8

39

-

94

8

-

12

8

78

-

106

8

-

14

8

156

-

97

9

-

14

5

313

-

81

6

-

14

4

1250

-

0

0

-

0

0

5000

-

0

0

-

0

0

Positive control

-

522

411

-

604

211

 

Table 3 Experiment 3 Mean number of revertants (average of 3 plates)

Concentration (µg/plate)

+/- S9 mix

TA 100

TA 1535

WP2uvrA

TA 98

TA 1537

Control

-

99

8

20

21

7

20

-

105

10

-

18

7

39

-

107

11

-

19

6

78

-

110

10

22

19

6

156

-

103

11

14

17

7

313

-

86

8

16

15

4

625

-

-

-

13

-

-

1250

-

0

0

0

0

0

2500

-

-

-

0

-

-

5000

-

0

0

0

0

0

Positive control

-

510

419

655

601

183

Control

+

127

10

21

23

8

156

+

130

9

29

26

9

313

+

132

12

17

19

7

625

+

127

12

22

18

6

1250

+

114

8

15

14

7

2500

+

119

6

13

6

3

5000

+

103

6

12

3

2

Positive control

+

679

304

758

517

152

 

Conclusions:
In a reliable study, conducted according to OECD guideline 471, no genotoxicity was seen in a bacterial mutagenicity assay. The study was performed in compliance with GLP.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
14-Jan-2003 to 17-Jan-2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Standards for mutagenicity tests using microorganisms (Notification no. 77, 1988, and Notification no. 67, 1997); Amendment of the reporting form of the results of the mutagenicity tests using microorganisms (Notification no. 653, 1997)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only duplicate plates used)
GLP compliance:
no
Remarks:
Japanese guideline notification no. 76
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital- and 5,6-benzoflavone-induced male rat liver S9
Test concentrations with justification for top dose:
10, 20, 39, 78, 156, 313, 625, 1250 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance supplied in water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
furylfuramide
Remarks:
-S9; TA100, WP2 uvrA, 0.01 µg/plate; TA98, 0.1 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
-S9; TA1535, 0.5 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ICR-191 (2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine.2HCl
Remarks:
-S9; TA1537, 1.0 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
+S9; TA100, TA98, TA1537, 5 µg/plate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
+S9; TA1535, 2 µg/plate; WP2 uvrA, 10 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: duplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition
Evaluation criteria:
"If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was judged positive."
Statistics:
Not done
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:
cytotoxicity
Remarks:
>=313 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=625 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: test substance supplied as a solution in water
- Precipitation: no precipitate seen
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
- Concentrations tested: 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate without S9
- Growth inhibition in all S. typhimurium TA strains at 313 µg/plate and above and in WP2 uvrA at 1250 µg/plate and above
- No precipitate
- Highest concentrations selected for the main test: 313 µg/plate for all S. typhimurium TA strains and 1250 µg/plate for WP2 uvrA
- Other concentrations obtained by 1:2 dilutions to provide 6 concentrations for each strain

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Table 1 Dose finding experiment: Revertants per plate (mean of two plates)

Concentration (µg/plate)

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0

134

128

23

14

19

27

18

39

16

23

1.2

137

131

21

13

20

30

19

35

16

26

4.9

127

142

18

16

24

26

16

33

17

22

20

114

127

28

16

20

34

18

32

19

17

78

132

130

18

13

29

30

18

31

14

22

313

46*

114

8*

8

21

18

11*

20

5*

12

1250

0*

125*

0*

4*

0*

17*

0*

10*

0*

4*

5000

0*

0*

0*

0*

0*

0*

0*

0*

0*

0*

Positive control

486

842

461

344

135

595

511

226

1767

82

* The growth inhibition of tested bacterium by the test substance was observed.

Table 2 Main experiment: Revertants per plate (mean of two plates)

Concentration (µg/plate)

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0

117

137

17

13

27

26

17

43

8

22

10

137

NT

21

NT

NT

NT

15

NT

9

NT

20

124

NT

15

NT

NT

NT

21

NT

11

NT

39

124

125

9

11

22

27

22

37

9

23

78

125

136

15

10

27

31

11

37

12

15

156

116

133

16

17

20

26

17

39

6*

13

313

50*

115

6*

9

16

19

11*

32

6*

12

625

NT

106*

NT

4*

10*

21

NT

23*

NT

5*

1250

NT

108*

NT

2*

0*

13*

NT

3*

NT

5*

Positive control

496

810

451

321

160

563

588

200

1739

69

* The growth inhibition of tested bacterium by the test substance was observed.

Conclusions:
In a reliable study, conducted to Japanese guidelines on mutagenicity tests (notification nos. 77 and 653), no genotoxicity was seen in a bacterial mutagenicity assay at up to 1250 µg/plate. The study was performed in compliance with Japanese guideline notification no. 76.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
29.07.1981 - 15.09.1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The deviation was that the number of strains used did not comply with the current guidelines.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four strains used
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
Spot test 25 µl, plate incorporation 0.001 µl, 0.004 µl, 0.01 µl, 0.02 µl, 0.04 µl, 0.1 µl, 0.2 µl, 0.3 µl, 1 µl, 3 µl, 10 µl (stock concentration 500 µl/ml; volume per plate 20µl)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: High purity water or DMSO
- Justification for choice of solvent/vehicle: none given in report.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA98 and TA100 with metabolic activation 5 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene at 5 µg/ml and 7 µg/ml
Remarks:
TA1535 and TA1537 with metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA98 and TA100 without metabolic activation 4 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium nitrite at 9 mg/ml
Remarks:
TA1535 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537 without metabolic activation 100 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate plates except for positive and negative controls where only one plate was tested, test not repeated


DETERMINATION OF CYTOTOXICITY
- Method: condition of background lawn


Evaluation criteria:
A positive response in the plate incorporation test is indicated if three or more treatments on the initial test (and/or retest, if performed) are significantly greater than solvent control (p=0.01) and if there is a significant positive dose response (p=0.01) for the initial test (and/or retest, if performed).
Statistics:
Bartlett's test
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:
cytotoxicity
Remarks:
10 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Average number of revertants per plate

Concentration

(µg/plate)

TA 98

TA 100

TA 1535

TA 1537

-S9

+ S9

-S9

+ S9

-S9

+ S9

-S9

+ S9

Solvent control

 32.0

 40.0

145.6

153.6

 13.3

13.6

  6.3

 12.3

Negative control

 26.0

 44.0

141.0

136

 23.0

12.0

  6.0

 19.0

10

  -

 27.6

  -

   6.6

  -

 3.0

   -

  3.0

3

  -

 39.0

  -

 -

  -

 4.0

   -

 16.0

1

 23.0

 39.6

  -

153.6

  2.3

 8.3

 4.6

 17.0

0.3

  20.6

 -

 90.3

 

 10.3

 -

 9.0

 -

0.2

  -

 51.3

  -

161.6

  -

10.0

  -

  8.0

0.1

  34.0

 -

153.0

 

 18.0

 -

 10.6

 -

0.04

  -

 51.5

  -

161.6

  -

12.0

  -

 11.3

0.02

  35.3

 -

166.3

 

 18.0

 -

  3.6

 -

0.01

  -

 47,6

  -

144.3

  -

10.0

  -

 12.0

0.004

 36.3

 -

144.6

 

 20.0

 -

 10.0

 -

0.001

 26.6

 -

162.3

 

 18.0

 -

 4.3

 -

Positive control

639.0

569.0

1214.0

1310.0

570.0

307.0

622.0

256.0

Conclusions:
EDTMP-xNa has been tested for mutagenicity to bacteria in a study, conducted according to a protocol that is similar to OECD Test Guideline 471, but prior to GLP. No evidence of mutagenicity to bacteria was found with or without microsomal activation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. Solvent, negative and positive control were included and gave the expected results.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The restrictions were that only four strains of bacteria were used.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only four strains of bacteria used
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
1-1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given in report
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 and TA 100 without metabolic activation: 4 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
TA 98 and TA 100 with metabolic activation: 5 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: NaNO2 9 µg/plate
Remarks:
TA 1535 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without metabolic activation: 75 µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminonthracene 5 µg/plate
Remarks:
TA 1535 and TA 1537 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); as impregnation on paper disk

DURATION

- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: initial spot plate test; triplicate plates in repeat plate incorporation study

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of background lawn (plate incorporation); zones of inhibition of growth (spot test)
Evaluation criteria:
A positive response is indicated if three or more treatments are significantly greater than solvent control (p=0.01) and if there is a significant positive dose-response (p=0.001).
Statistics:
Treatment compared with solvent controls using a one-sided t-test and within-levels pooled variance
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:
cytotoxicity
Remarks:
1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Spot test: toxicity but no mutagenicity was observed in strains TA 98 and TA 100. No toxicity or mutagenicity was observed in strains TA 1535 and TA 1537.

Number of revertants per plate

Concentration

(µg//plate)

TA 98

TA 100

TA 1535

TA 1537

+ S9

   

+ S9

+ S9

   

+ S9

Solvent control

40

16

22

146

134

150

7

11

10

2

8

3

Negative control

41

 -

 -

170

 -

 -

13

 -

 -

9

 -

 -

10

22

36

39

95

93

96

8

6

4

5

12

6

 3

22

14

33

99

70

100

6

6

4

7

3

6

 1

38

35

23

128

134

102

5

5

6

6

7

4

 0.2

37

40

30

145

149

139

5

5

10

7

6

2

 0.04

42

55

36

142

152

165

8

5

6

7

6

5

 0.01

31

29

57

128

135

142

11

7

8

5

6

6

Positive control

495

 -

 -

1078

 -

 -

178

-

-

166

-

-

 

 

Concentration

(µg//plate)

TA 98

TA 100

TA 1535

TA 1537

- S9

- S9

- S9

- S9

Solvent control

18

22

11

137

104

136

4

3

4

2

5

2

Negative control

22

 -

 -

150

 -

 -

11

-

-

6

-

-

1

12

12

5

150

121

142

4

3

4

2

5

2

0.3

13

20

17

141

123

123

7

12

5

3

4

7

0.1

22

26

13

135

136

104

6

4

8

5

4

2

0.2

20

16

15

181

153

141

9

6

7

8

5

4

0.004

27

15

19

150

136

139

4

5

12

8

5

2

0.001

20

17

23

94

107

97

3

10

10

4

5

4

Positive control

378

 -

 -

669

 -

 -

198

 -

 -

125

 -

 -

 

 

Conclusions:
EDTMP-Ca-Na salt has been tested for mutagenicity to bacteria in a study, conducted according to a protocol that is similar to OECD Test Guideline 471, but prior to GLP. No increase in the number of revertants was observed when Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were tested up to cytotoxic concentrations with and without metabolic activation. Appropriate positive, negative and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The restrictions were that only a short exposure time was used.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
short exposure time (1 cell cycle)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F12 with 5% newborn calf serum
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
10-50 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: growth medium
- Justification for choice of solvent/vehicle:: none given in report
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3, 6 and 12 hours


SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: Two different Lots, A and B were treated. The experiment with Lot A was repeated because the positive control gave a negative result. Lot A was tested with and without sodium fluoride, Lot B with and without metabolic activation.

NUMBER OF CELLS EVALUATED: 100 per culture per Lot

VALUATION: cells were evaluated for chromatid and chromosome deletions and exchanges.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:
A result is considered positive if it is statistically higher than that of the solvent control (p/=0.05).
Statistics:
T-test used for comparison between treated and solvent control results.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1 summary of in vitro cytogenicity study: Lot A

Treatment

% aberrant cells

Mitotic Index

-NaF

+NaF

-NaF

+NaF

Initial experiment, 3 h harvest

0*

4

3

0.06

0.05

30

0

1

0.03

0.04

40

0

1

0.06

0.04

50

0

0

0.04

0.04

 

Initial experiment, 6h harvest

0*

0

1

0.08

0.06

30

0

0

0.04

0.05

40

0

0

0.06

0.04

50

0

0

0.05

0.05

 

Initial experiment, 12h harvest

0*

0

2

0.07

0.10

30

1

1

0.07

0.08

40

1

3

0.05

0.04

50

1

2

0.04

0.05

Positive control

1

1

NR

NR

 

repeat experiment, 12h harvest

0*

4

6

0.08

0.09

30

4

3

0.07

0.07

40

4

3

0.04

0.04

50

3

7

0.04

0.03

Positive control

31

49

NR

NR

* Solvent control with growth medium

NR: not recorded

 

Table 2 summary of in vitro cytogenicity study: Lot B

Treatment

% aberrant cells

Mitotic Index

-MA

+MA

-MA

+MA

3 h harvest

0*

1

0

0.11

0.06

100

1

10

0.07

0.04

200

0

4

0.07

0.06

500

1

2

0.10

0.05

 

6h harvest

0*

1

6

0.05

0.05

100

1

7

0.05

0.04

200

1

4

0.03

0.05

500

0

1

0.05

0.05

 

12h harvest

0*

0

6

0.9

0.07

100

0

6

0.01

0.02

200

2

7

0.01

0.01

500

1

5

0.01

0.01

Positive control

7

14

NR

NR

* Solvent control with growth medium

NR: not recorded

Conclusions:
EDTMP-H, has been tested for clastogenicity, according to protocol that is similar to OECD Test Guideline 473 and in compliance with GLP. No test substance-induced increase in the incidence of chromosome aberrations in Chinese hamster ovary cells was observed with or without metabolic activation using either Lot A or Lot B. The substance was concluded to be negative for the induction of chromosome aberrations. Appropriate solvent and positive controls were included and gave expected results.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2012-07-18 to 2012-07-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Only one strain of bacteria was tested.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only one strain of bacteria tested
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
only one strain of bacteria tested
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella.
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation: methyl methane sulfonate, MMS used with Strain: TA 102 @ 3 µL/plate
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates. Initial plate incorporation assay was repeated using the pre-incubation method.


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is prepared from 8 – 12 weeks old male Wistar rats (Hsd Cpb: WU; weight approx. 220 – 320 g, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 39.5 mg/ml (lot no. R 260412) in both experiments.

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.



Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
number of revertants was reduced though background lawn condition was normal
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No precipitation of the test item occurred up to the highest investigated dose.

COMPARISON WITH HISTORICAL CONTROL DATA: performed

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate in both independent experiments



Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed at the following concentrations (µg/plate):

Strain Experiment I Experiment II
without S9 mix with S9 mix without S9 mix with S9 mix
TA 102 1000 - 5000 333 - 5000 2500, 5000 100 - 5000

Summary of Results Pre-Experiment and Experiment I Plate incorporation revertants per plate mean of three plates

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

Without Activation

With Activation

Solvent control (water)

 

355 ± 30

469 ± 21

Untreated

 

330 ± 6

497 ± 97

EDTMP, pH-neutral

3 µg

357 ± 26

498 ± 43

10 µg

367 ± 6

446 ± 45

 

33 µg

371 ± 17

430 ± 10

 

100 µg

354 ± 22

263 ± 23

 

333 µg

235 ± 24

35 ± 19

 

1000 µg

10 ± 8

3 ± 3

 

2500 µg

0 ± 1

0 ± 0

 

5000 µg

0 ± 0

0 ± 0

Positive control

3.0 µl

2637 ± 381

1824 ± 268

Concentrations expressed as active acid content, Correction factor 1.25

Summary of Results Experiment 2 Pre-incubation revertants per plate mean of three plates

Test

Group

Dose Level

(per plate)

Revertant Colony Counts (Mean ±SD)

Without Activation

With Activation

Solvent control (water)

 

393 ± 28

554 ± 42

Untreated

 

384 ± 4

586 ± 9

EDTMP pH-neutral

3 µg

406 ± 22

626 ± 33

10 µg

414 ± 7

613 ± 59

 

33 µg

394 ± 19

552 ± 39

 

100 µg

365 ± 26

231 ± 36

 

333 µg

412 ± 12

33 ± 16

 

1000 µg

369 ± 27

4 ± 2

 

2500 µg

15 ± 2

2 ± 4

 

5000 µg

1 ± 1

2 ± 3

Positive control

3.0 µl

3432 ± 58

1743 ± 284

Test substance concentrations expressed as active acid content, Correction factor 1.25

Conclusions:
EDTMP-xNa has been tested for mutagenicity to bacteria in a study, conducted according to OECD Test Guideline 471 and in compliance with GLP. No increase in the number of revertants was observed with or without activation in either the initial plate incorporation assay or the repeat experiment using pre-incubation in Salmonella typhimurium strain TA 102. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Executive summary:

The test item EDTMP, pH-neutral was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strain TA 102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without S9 mix in both independent experiments..

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 102

1000 - 5000

333 - 5000

2500, 5000

100 - 5000

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with EDTMP, pH-neutral at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: The study did not meet current guideline requirements for bacterial mutagenicity, and only a summary report was available for review. It does, however, add weight of evidence for bacterial mutagenicity.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
range of strains does not comply with current guidance. Full details of method are not included. Tested only up to 1000 µg/plate, no toxicity observed, apparently no replications
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
mammalian metabolic activation
Test concentrations with justification for top dose:
0.1-1000 µg/plate
Vehicle / solvent:
no information
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Species / strain:
S. typhimurium, other: not specified
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

Table 1 number of revertants per plate

Concentration µg

TA 98

TA 100

TA 1535

TA 1537

TA 1538

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0.1

16

37

43

48

20

17

3

9

13

39

1

14

43

58

56

15

11

6

4

6

34

10

18

45

56

57

20

9

7

15

7

37

100

16

50

59

71

15

6

6

20

5

47

500

17

44

55

60

17

7

10

17

7

35

1000

21

34

95

56

14

8

10

13

4

29

Solvent control

20

40

68

63

29

15

4

6

7

29

Positive control

14

170

87

>3000

+

+

2548

1248

20

>3000

Conclusions:
HMDTMP-acid has been tested according to a protocol this is similar to OECD 471. Full details are not included in the summary report available. No evidence of mutagenicity was observed at any concentration in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other:
Remarks:
The study did not meet current guideline requirements for bacterial mutagenicity, and only a summary report was available for review. It does, however, add weight of evidence for bacterial mutagenicity.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only two strains of bacteria used
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium, other: TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
100, 500, 1000, 5000, 10000 and 40000 µg/plate, equivalent to 25, 125, 250, 1250, 2500 and 10 000 µg active acid per plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: none given
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-amino anthracene 2 µg
Remarks:
TA 98 and TA 100, with and without metabolic activation
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
TA 100 without metabolic activation 670 µg
Untreated negative controls:
yes
Remarks:
Untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Daunomycin
Remarks:
TA 98 without metabolic activation
Details on test system and experimental conditions:
ACTIVATION: S9 mix contained glucose-6-phosphate and NADP as co-factors. 0.1 ml of S9 mix containing 30% S9 was added to 2 ml top agar, 0.1 ml bacterial suspension and 0.1 ml of test solution, giving a concentration of S9 of approximately 1%.

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: triplicate plates

DETERMINATION OF CYTOTOXICITY: none recorded

Evaluation criteria:
Results were considered negative if the plates exposed to test compound were similar to the solvent controls.
Species / strain:
S. typhimurium, other: 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

Table 1 number of revertants per plate (triplicate plates)

Concentration µg/plate

TA 100

TA 98

-MA

+MA

-MA

+MA

Untreated

72, 51 56

63, 75, 60

14, 17, 21

10, 9, 6

Solvent control

65, 53, 54

66 94, 79

12, 20, 15

7, 8, 9

100

71, 83, 80

62, 60, 83

7, 11, 9

9, 12, 9

500

57, 57, 67

88, 79, 68

11, 11, 10

11, 8, 6

1000

61, 73, 55

62, 62, 76

12, 8, 4

12, 7, 10

5000

74, 79, 80

67, 70, 51

9, 11, 11

4, 9, 6

10 000

87, 63, 81

71, 57, 58

7, 9, 4

8, 6, 7

31 300

75, 70, 73

81, 72, 74

21, 11, 7

8, 8, 8

2-aminoanhracene

59, 56, 63

834, 808, 788

1, 4, 7

370, 280, 281

methylmethane sulphonate

780, 788, 823

NT

NT

NT

Daunomycin 5 µg/plate

0.5 µg/plate

NT

NT

NT

NT

59, 76, 64

50, 45, 34

NT

NT

NT not tested

Conclusions:
HMDTMP-xNa has been tested according to a protocol that is similar to OECD 471, using Salmonella typhimurium strains TA 98 and TA 100. No increase in the number of revertants was observed at any concentration with and without metabolic activation. Appropriate untreated, solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo chromosome aberration: read-across from DTPMP-H, negative (similar to OECD Test Guideline 475) (Hazleton Laboratories, 1983)

In vivo chromosome aberration: read-across from EDTMP-H, negative (similar to OECD Test Guideline 475) (EG&G, 1981)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15-Aug-1983 to 04-Nov-1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
There is some inaccuracy with respect to test substance identity.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
(insufficient cells scored for aberrations and for mitotic index)
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, New York, USA
- Age at study initiation: 64 days
- Weight at study initiation: 275-286 g males, 198-204 g females
- Assigned to test groups randomly: yes, via computer-generated random numbers
- Fasting period before study: no data
- Housing: individually in wire mesh cages
- Diet (e.g. ad libitum): Purina Lab Meal #5001, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 74-83
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 15-Aug-1983 To: 17-Aug-1983
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: no data, but well-known vehicle
- Concentration of test material in vehicle: 20, 66 and 197 mg active acid/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
- Purity: distilled
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Freshly prepared on the day of administration
Duration of treatment / exposure:
Single oral gavage dose; post-treatment sampling times were 6, 12, 24 and 48 hours
Frequency of treatment:
Once
Post exposure period:
6, 12, 24 and 48 hours
Dose / conc.:
200 other: mg active acid/kg bw
Remarks:
Calculated from nominal concentration of test substance
Dose / conc.:
660 other: mg active acid/kg bw
Remarks:
Calculated from nominal concentration of test substance
Dose / conc.:
1 970 other: mg active acid/kg bw
Remarks:
Calculated from nominal concentration of test substance
No. of animals per sex per dose:
6
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data, but well-known clastogen
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Selected on the basis of a range-finding test in which no effect on mortality or mitotic index and minimal clinical signs were observed at the top dose of 1970 mg active acid/kg bw only; all 3 dose levels analysed.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Colchicine administered 4, 10, 22 and 46 hours after treatment; animals sacrificed 2 hours later.

DETAILS OF SLIDE PREPARATION: Bone marrow cells collected from femurs by aspiration into Hank's Balanced Salt Solution; after centrifugation, supernatant decanted and cells suspended in warm 0.075 M KCl for 25 minutes; cells fixed using 3:1 methanol:acetic acid fixative; chilled then dispersed on glass microscope slides (2/animal) and air dried; stained with Giemsa and mounted with glass coverslips.

METHOD OF ANALYSIS: Slides coded; attempted to examine >=60 metaphases from 5/6 rats per sex per group (if not possible, 6th animal also analysed); 48-hour timepoint not analysed; for each animal, data recorded included numbers and types of chromosome aberrations, mitotic index (500 cells/animal), modal number for each metaphase; chromosome aberrations were classified as chromatid breaks, chromosome breaks, chromatid and chromosome gaps, exchanges, cells with >10 aberrations, pulverized cells.
Evaluation criteria:
No data
Statistics:
Mean mitotic index, mean modal number, percent aberrant cells and mean number of aberrations per cell analysed by Kruskall-Wallis non-parametric non-parametric analysis of variance and non-parametric pairwise group comparisons. Body weight analysed by analysis of covariance. All tests one-tailed.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
body weight loss, clinical observations
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 200, 660, 1970 mg active acid/kg bw
- Solubility: miscible with water
- Clinical signs of toxicity in test animals: no mortality; "a few abnormal clinical observations were observed at the highest dose"
- Evidence of cytotoxicity in tissue analyzed: no effect on mitotic index
- Rationale for exposure: slight toxicity at the highest dose
- Harvest times: observed 1 day after dosing
- High dose with and without activation: not applicable
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): none
- Appropriateness of dose levels and route: no data
- Statistical evaluation:
- no statistically significant increase in chromosome damage at any dose at any timepoint in either sex; positive control (24 hours) statistically significantly incresased
- no statistically significant change in mitotic index
- no statistically significant change in mean modal number

3/12 animals died when treated with 1970mg/kg.  Mild clinical signs seen at this dose.  Loss of body weight in top dose animals (both sexes) over 48 hours observed. No evidence of mitotic delay so 48 hour animals not analyzed.

Table 1 Results of Chromosome aberration study

Treatment (mg/kg bw)

Treatment time (hrs)

Number of animals analyzed per group

Number of cells analyzed

% aberrant cells per group

Control

6

12

600

0.5

200

6

11

600

0

660

6

11

600

0.3

1970

6

11

600

0.5

Control

12

10

600

0

200

12

11

600

0

660

12

11

561

0.2

1970

12

10**

600

0.2

Control

24

11

540

0

Positive control

24

12

280

7.9

200

24

11

540

0.2

660

24

11

540

0

1970

24

10**

540

0.6

** Animals found dead prior to sacrifice

Conclusions:
In a reliable study, conducted using a protocol similar to OECD guideline 475, no evidence of clastogenicity was seen in rat bone marrow following a single oral gavage administration at doses up to the maximum tolerated dose of 1970 mg active acid/kg bw. The study was performed in compliance with GLP.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May 08, 1981 to June 22, 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The restrictions were that only 50 cells per animal were evaluated, and samples were collected only once. The study was conducted in compliance with GLP.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
Only 50 cells/animal were evaluated instead of 100 cells. Animals were dosed for consecutive 5 days instead of single treatment and justification for same was not provided. Samples were collected only once instead of at least 2 times.
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Outbred albino rats were obtained from Taconic farms
- Age at study initiation: Sexually mature
- Weight at study initiation: 125-200 g
- Assigned to test groups randomly: Yes, animals were randomized by weight
- Fasting period before study: Not reported
- Housing: Animals were housed individually.
- Diet: Food (Agway RMH 3000), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 7-9 d prior to treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 69-75°F (mean)
- Humidity: 49-73% (mean)
- Air changes: Not reported
- Photoperiod: Not reported

IN-LIFE DATES: Not reported
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solution of test substance was prepared in corn oil. The dosing solution was prepared freshly prior to treatment.

DOSE VOLUME: 1 ml/100 g bw
Duration of treatment / exposure:
5d
Frequency of treatment:
Once daily
Post exposure period:
22-24 h after last treatment
Dose / conc.:
240 mg/kg bw/day
Dose / conc.:
800 mg/kg bw/day
Dose / conc.:
2 400 mg/kg bw/day
No. of animals per sex per dose:
5 rats/sex/ group
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
- Positive control: Methylmethane sulfonate (MMS)
- Solvent used: Distilled water
- Route of administration: Oral gavage
- Doses / concentrations: 0.156 g/ kg bw/ d
- Dose volume: 1.5 ml/ 150 g bw
- Duration of dosing: Once daily for 5 d
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
SACRIFICE: Animals were sacrificed by cervical dislocation following CO2 anaesthesia after 2-4 h treatment with Colchicine.

TREATMENT AND SAMPLING: Animals were treated once daily for 5 consecutive days. Animals were treated with Colchicine (intraperitoneal; 1 mg/ kg) approx. 20 h after last treatment, to cause mitotic arrest of bone marrow cells. Bone marrow cells were collected 2-4 h after treatment with Colchicine. Bone marrow of both femurs was aspirated into 10cc syringe filled with prewarmed (37°C) 5 mL Hank’s balanced salt solution. After centrifugation (100x g), cells were suspended in 0.075 M KCl for 10 min at 37±1°C.

DETAILS OF SLIDE PREPARATION AND STAINING: 2-3 drops of cell suspension were placed onto microscopic slides and passed through flame. All slides were stained with Giemsa stain for 8 min and fixed with Carnoy’s fixative. Slides were washed in water (1 min) then with acetone (10-20 sec), then in acetone-xylene (1:1 v/ v; 10-20 sec) and finally in xylene for 5 min. Permount was used as the mounting and all slides were placed on slide warmer at 30-35°C for minimum 2 h before scoring.

METHOD OF ANALYSIS: 50 metaphase/ animal were analyzed. Cytogenetic abnormalities such as deletions, exchanges, rings, gaps and breaks were scored and mitotic index of each animal was determined. The vernier location for each cell scored was recorded.
Statistics:
Standard deviation (SD) and standard error (SE) was calculated.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

DETAILS ON CLINICAL SIGNS AND SYMPTOMS:

- Clinical signs: Animals treated with test substance revealed no signs of toxicity.

- Mortality: No mortality was observed in negative, vehicle and test substance treated groups.

RESULTS OF POSITIVE CONTROL:

- Clinical signs: Some of positive control group animals revealed signs of ataxia, inactivity, lethargy, weakness and hypersensitivity.

- Mortality: 4 animals of positive control group died during study.

Table 1. Bone marrow aberrations after treatment with Ethylenediamine tetraphosphoric acid and controls (Study # 25691)

Treatment

% of Total Cells Analyzed

Total aberrations (including gaps)

Aberrations excluding gaps

Male

Female

Male

Female

Distilled water

0.6

2.8

0

0.4

Corn oil

4.0

7.2

0

1.6

MMS(0.156 g/ kg bw)

18.0

38.5

9.0

15.4

Test substance

(0.24 g/ kg bw)

3.6

4.4

0

0.4

Test substance

(0.80 g/ kg bw)

2.0

4.8

0.4

0.8

Test substance

(2.40 g/ kg bw)

4.0

2.8

0.4

0

MMS= Methylmethane sulfonate

Conclusions:
EDTMP-H has been tested in a chromosome aberration assay conducted according to a protocol that is similar to OECD 475 and in compliance with GLP conditions. The substance was administered orally at 0.24, 0.80 and 2.40 g/kg bw/d to albino Sprague Dawley rats for 5 days. No test substance treatment related mortalities were observed. Appropriate solvent (corn oil), negative (water) and positive controls were included and gave expected results. The test substance did not induce any chromosomal damage in the bone marrow cells of the treated rats. It is concluded that EDTMP-H is negative for the induction of chromosome aberrations in rat bone marrow under the conditions of the study.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

There are no genetic toxicity data available for HMDTMP (4-7Na).

Data from other category members within the HMDTMP category are used for the following endpoints: Gene mutation (Bacterial reverse mutation assay / Ames test) and mutagenicity to mammalian cells. See attachment to Section 13 for justification of read-across within HMDTMP Category.

Data from EDTMP Category are used for the following endpoints: Bacterial mutagenicity in vitro; cytogenicity in mammalian cells in vitro and in vivo; mammalian mutagenicity in vitro. See attachment to Section 13 for justification of read-across.

Data from DTPMP Category are used for the following endpoints: Bacterial mutagenicity in vitro; cytogenicity in mammalian cells in vitro and in vivo; mammalian mutagenicity in vitro. See attachment to Section 13 for justification of read-across.

HMDTMP-xNa has been tested in bacterial reverse mutation assay, according to a protocol that is similar to OECD Test Guideline 471, but not in compliance with GLP. The substance was tested on Salmonella typhimurium strains TA 98 and TA 100. No increase in the number of revertants was observed at any concentration with and without metabolic activation. Appropriate untreated, solvent and positive controls were included and gave expected results (Department of Biochemistry, University of Birmingham, 1981).

HMDTMP-acid has been tested in bacterial reverse mutation assay, according to a protocol that is similar to OECD Test Guideline 471, but prior to GLP. Full details are not included in the summary report available. No evidence of mutagenicity was observed at any concentration in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 with and without metabolic activation. Appropriate solvent and positive controls were included and gave expected results (Monsanto, 1976).

No data are available for HMDTMP for mutagenicity to a bacterial strain capable of detecting cross-linking or oxidising mutagens. In view of the lack of genetic toxicity demonstrated in studies on mammalian cells, and the absence of structural features that indicate that such mutagenicity is likely, testing in an appropriate 5th strain is not considered necessary. In addition, the results from genetic toxicity assays on substances in the aminomethylenephosphonates analogue group do not indicate that these substances are mutagenic, and include bacterial mutagenicity studies using an appropriate 5th strain studies on DTPMP Category members and EDTMP Category members. All these results were negative.

The structural analogue, DTPMP-H, has been tested in bacterial reverse mutation assay, conducted to Japanese guidelines on mutagenicity tests (notification nos. 77 and 653). No genotoxicity was seen in a bacterial mutagenicity assay for DTPMP-H at concentrations up to 1250 µg/plate when tested with or without metabolic activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (BMI Inc, 2003).

The structural analogue, DTPMP-7Na, has been tested in bacterial reverse mutation assay, conducted according to a protocol similar to OECD Test Guideline 471 and in compliance with GLP. In the study an aqueous solution containing DTPMP-7Na (pH 6-8) was tested for the induction of gene mutations in Salmonella typhimurium strains TA98, 100, 1537 and 1535 and in Escherichia coli strain WP2 uvrA (Japan Oilstuff Inspectors Corporation, 2001). The study was conducted using the preincubation modification in the presence and absence of S9 mix on two occasions. The test substance did not induce mutations in either strain at concentrations up to 5000 µg/plate, the accepted upper limit for this assay.

The structural analogue, EDTMP-xNa has been tested for mutagenicity to bacteria in a study, conducted according to a protocol that is similar to OECD Test Guideline 471, but prior to GLP. No evidence of mutagenicity to bacteria was found with or without microsomal activation in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. Solvent, negative and positive control were included and gave the expected results (Monsanto, 1981a).

The structural analogue, EDTMP-Ca-Na salt has been tested for mutagenicity to bacteria in a study, conducted according to a protocol that is similar to OECD Test Guideline 471, but prior to GLP. No increase in the number of revertants was observed when Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 were tested up to cytotoxic concentrations with and without metabolic activation. Appropriate positive, negative and solvent controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Monsanto, 1981b).

The structural analogue, EDTMP-xNa has been tested for mutagenicity to bacteria in a study, conducted according to OECD Test Guideline 471 and in compliance with GLP. No increase in the number of revertants was observed with or without activation in either the initial plate incorporation assay or the repeat experiment using pre-incubation in Salmonella typhimurium strain TA 102. Appropriate positive, solvent and untreated controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test (Harlan Cytotest Cell Research GmbH, 2012)

The structural analogue, EDTMP-H, has been tested for clastogenicity, according to protocol that is similar to OECD Test Guideline 473 and in compliance with GLP. No test substance-induced increase in the incidence of chromosome aberrations in Chinese hamster ovary cells was observed with or without metabolic activation using either Lot A or Lot B. The substance was concluded to be negative for the induction of chromosome aberrations. Appropriate solvent and positive controls were included and gave expected results (Monsanto, 1986a).

The structural analogue, DTPMP-7Na, has been tested for clastogenicity in a reliable study, conducted according to OECD Test Guideline 473 and in compliance with GLP. A dose related increase in the number of cells with aberrations was observed after 48 hours treatment in an in vitro chromosome aberration assay. The substance was concluded to be positive for the induction of chromosome aberrations. Appropriate solvent and positive controls were included and gave expected results (Japan Oilstuff Inspectors Corporation, 2001).

To follow-up on the positive result for DTPMP-7Na, a comet assay is going to be conducted. The study result will be added when available.

HMDTMP (4-7K) has been tested for mutagenicity to mammalian cells according to a protocol that is similar to OECD Test Guideline 476, but prior to GLP. No increase in the mutant frequency was observed at any concentration up to cytotoxic concentrations, with and without metabolic activation. Appropriate positive, solvent and negative controls were included and gave the expected results. It is concluded that the substance is negative for mutagenicity to L5178Y mouse lymphoma cells under the conditions of the test. (Litton Bionetics, 1978).

The structural analogue, DTPMP-xNa, has been tested for mutagenicity to mammalian cells in a reliable study, conducted according to OECD Test Guideline 476 and in compliance with GLP. No genotoxicity was seen in an in vitro mouse lymphoma L5178Y cells when tested in the presence or absence of metabolic activation. However, the highest concentration selected for testing was below the maximum required concentration by the guideline. Appropriate positive and solvent controls were included and gave the expected results (Central Toxicology Laboratory, 1997).

The structural analogue, DTPMP-H, has been tested for mutagenicity to mammalian cells in a reliable study, conducted according to a protocol similar to the OECD Test Guideline 476 and in compliance with GLP. No evidence of mutagenic potential was detected in Chinese hamster ovary cells in vitro in the absence or presence of an exogenous metabolic activation system in an assay evaluating mutation at the HGPRT locus. Appropriate positive and negative controls were included and gave the expected results (Pharmakon Research International, 1984).

The structural analogue, EDTMP-xNa, has been tested in CHO cells for its potential to induce forward mutation in a study, conducted according to a protocol that is similar to OECD Test Guideline 476 and in compliance with GLP.  No evidence of mutagenicity was observed with or without metabolic activation in the initial or the repeat experiments. Appropriate positive and test medium controls were included and gave expected results.  It is concluded that the test substance is negative for induction of forward mutations in CHO cells under the conditions of the test (Monsanto, 1986b).

The structural analogue, DTPMP-H, has been tested in a chromosome aberration assay conducted according to a protocol that is similar to OECD Test Guideline 475 and in compliance with GLP. No evidence of clastogenicity was seen in rat bone marrow following a single oral gavage administration at doses up to the maximum tolerated dose of 1970 mg active acid/kg bw. Appropriate solvent and positive controls were included and gave expected results (Hazleton Laboratories, 1983).

The structural analogue, EDTMP-H, has been tested in a chromosome aberration assay conducted according to a protocol that is similar to OECD Test Guideline 475 and in compliance with GLP. The substance was administered orally at 0.24, 0.80 and 2.40 g/kg bw/d to albino Sprague Dawley rats for 5 days. No test substance treatment related mortalities were observed. Appropriate solvent (corn oil), negative (water) and positive controls were included and gave expected results. The test substance did not induce any chromosomal damage in the bone marrow cells of the treated rats. It is concluded that EDTMP-H is negative for the induction of chromosome aberrations in rat bone marrow under the conditions of the study (EG&G, 1981).

Justification for classification or non-classification

Based on the available in vitro genotoxicity data, no classification is required for mutagenicity for HMDTMP (4 -7Na) according to Regulation (EC) No 1272/2008.