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 analogous substance HMDTMP-xNa: negative with and without activation in Salmonella typhimurium strains TA98 and TA100 (similar to OECD 471) (Manley A (1981)).
Gene mutation (Bacterial reverse mutation assay / Ames test): read-across from analogous substance 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 471) (Flowers L (1976)).
Cytogenicity in mammalian cells: read-across from closely-related substance EDTMP-H: negative with and without metabolic activation in CHO cells (similar to OECD TG 473) (Li (1986)).
Mutagenicity in mammalian cells: negative with and without metabolic activation in L5178Y mouse lymphoma cells (similar to OECD TG 476) (Matheson D.W. 1978)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
DTPMP acid and DTPMP-7Na as well as HMDTMP (4-7K) are members of the aminomethylenephosphonates analogue group. See attachment for justification of read-across.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Remarks:
r/a from DTPMP-7Na
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
Remarks:
r/a from DTPMP-7Na
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
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Remarks:
r/a from DTPMP-H
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
Remarks:
r/a from DTPMP-H
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
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
EDTMP acid and HMDTMP (4-7K) are both members of the aminomethylenepphosphonates analogue group. See attachment for justification of read-across.
Reason / purpose:
read-across source
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
Endpoint:
genetic toxicity in vitro, other
Remarks:
mutagenicity in bacterial cells; mutagenicity in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
See justification for read-across between HMDTMP category members in the attached file.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Species / strain:
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
Species / strain:
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
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
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:
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:
HDDTMP-xK 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:
key study
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:
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 according to protocol that is similar to OECD 473. No test substance induced increase in the incidence of chromosome aberrations was observed with or without metabolic activation in either Lot A or Lot B. Appropriate solvent and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosomal aberrations under the conditions of the test.
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only 2-AA used as positive control +S9)
Qualifier:
according to
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:
Posltive: mean number of revertant colonies more than double the negatlve 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:
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:
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
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:
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:
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:
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:
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:
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

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Read-across within the HMDTMP category is used for the following endpoints: Gene mutation (Bacterial reverse mutation assay / Ames test) and Mutagenicity in mammalian cells. Read-across between members of the HMDTMP category is considered scientifically justified, as the counterions potassium and sodium are not significant in genotoxicity and have been assessed in depth in the public literature.

Read-across from EDTMP-H is used for the following endpoint: Cytogenicity in mammalian cells. Read-across between EDTMP-H and members of the HMDTMP category is considered justified as both EDTMP and HMDTMP are members of the same analogue group. See attached justification for read-across.

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 (Manley A (1981)).

HMDTMP-acid has been tested according to a protocol that 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 (Flowers L (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-acid and DTPMP-xNa. All these results were negative.

EDTMP-H has been tested according to protocol that is similar to OECD 473. 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. Appropriate solvent and positive controls were included and gave expected results (Li, A.P., Myers, C. A. (1986)).

HMDTMP-xK 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. (Matheson D.W. (1978))

Justification for classification or non-classification

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