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): negative with and without activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (OECD TG 471)
Cytogenicity in mammalian cells: positive in Chinese hamster lung IU cells (OECD TG 473)
Mutagenicity in mammalian cells: negative in L5278Y cells (similar to OECD TG 476)

Link to relevant study records

Referenceopen allclose all

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
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:
>= 625 µg mixed sodium salts/plate -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
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 cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
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:
In a reliable study, conducted according to OECD guideline 473, 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 study was performed in compliance with GLP.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-Jan-1997 to 25-Feb-1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test substance was not tested up to the maximum concentration required by guideline.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
(not tested to maximum concentration required by guideline)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI-1640
- Properly maintained: no data
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital- and beta-naphthoflavone-induced male rat liver S9
Test concentrations with justification for top dose:
Phase 1: 266-2128 µg active acid/ml
Phase 2: 265-2121 µg active acid/ml
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
Remarks:
without S9; 750 µg active acid/ml
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
with S9; 3 µg active acid/ml
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): no data

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: duplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: other: cell growth
Evaluation criteria:
- Criteria for a positive response: a statistically significant, dose related increase in mutant frequency is required, but not only at concentrations eliciting excessive toxicity. An associated absolute increase in mutant number above the solvent control values is a further requirement. Such a response must be reproducible in an independent experiment for the test substance to be described as positive in this assay.
- CrIteria for a negative response: A negative response is obtained when there is no reproducible statistically significant dose related increase in mutant frequency. When reproducible significant increases in mutant frequency are seen only at levels of excessive toxicity, or when such increases are not accompanied by an increase in absolute numbers of mutants over solvent control values, then the effect is considered not to be indicative of a positive response in this assay.
Statistics:
- If considered necessary, the data are considered by logit regression, using a complimentary log-log link function. The dependent variable is the number of empty wells. This procedure provided maximum likelihood estimates of log mutant frequencies. Variances are inflated by the between duplicate heterogeneity factor.
- Tests for trend in log mutant frequency with actual concentration are obtained separately for each experiment, in the presence and absence of S9-mix. In addition, an overall test for trend combining data across experiments is performed.
- Intergroup comparisons of log mutant frequency comparing each treated group with the solvent control are performed within each experiment. In addition, tests for trend in log mutant frequency with actual concentration are obtained both within each experiment and combined across all experiments.
- All tests are one-sided. Similar analyses are carried out separately for the positive controls.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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: test solutions neutralised to ~pH7
- Effects of osmolality: "higher concentrations [than 2200 µg mixed sodium salts/ml] produced excessive increases (up to 70 mM/kg) in the osmolality of the treatment medium. Such increases in osmolality have been associated with artefactual responses in genotoxicity assays ... and these concentrations were therefore considered not to be appropriate for testing."
- Evaporation from medium: no data
- Water solubility: soluble in water
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES: no data

COMPARISON WITH HISTORICAL CONTROL DATA: no data


ADDITIONAL INFORMATION ON CYTOTOXICITY:

No increases in mutant frequency were seen in any of the tests and it was not considered necessary to conduct statistical analysis.  All the positive control cultures had elevated mutant frequencies as expected.

The maximum concentration tested was 2200
µg/ml.  Higher concentrations were claimed to give excessively high osmolarity, although the values given for 4256 and 4242µg/ml in subsequent tests only resulted in increases to 354 and 334 mOsm/kg respectively. Since no increases in mutant frequency were seen in the first test at a dose producing 330 mOsm/kg it could be argued that the dose of 4242 could have been tested.  All concentrations below 5000 µg/ml are <10 mM, the upper limit defined by OECD for this assay.  A toxicity limit was not reached in these tests - top levels had >75% survival.  Therefore the upper limit defined for this assay was not reached.

Conclusions:
In a reliable study, conducted according to OECD guideline 476, no genotoxicity was seen in an in vitro mouse lymphoma L5178Y cell mutagenicity assay in the presence or absence of S9. However, the highest concentration selected for testing was below the maximum required by the guideline. The study was performed in compliance with GLP.
Endpoint:
in vitro gene mutation 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:
DTPMP xNa is a member of the DTPMP phosphonates category. The use of read-across data between members of the category is in accordance with the rationale outlined in the attached justification and Section 1.4 of the CSR.
Reason / purpose:
read-across source
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Bone marrow chromosome study in rats (oral gavage administration): Read-across from DTPMP acid: Negative (similar to OECD TG 475)

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:
key study
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:
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 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 chromsome 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 / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
DTPMP acid is a member of the DTPMP phosphonates category. The use of read-across data between members of the category is in accordance with the rationale outlined in the attached justification and Section 1.4 of the CSR.
Reason / purpose:
read-across source
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
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Information is available for DTPMP-xNa from bacterial and mammalian mutagenicity studies as well as an in vitro chromosome aberration study. An in vivo chromosome aberration study in rats with the category member, DTPMP-acid was also available. The use of read-across data between members of the category is in accordance with the rationale outlined in the toxicological information endpoint summary of the IUCLID and Section 1.4 of the CSR.

Bacterial mutagenicity

The result of the bacterial mutagenicity study was negative.

In vitro cytogenicity

In a reliable study, conducted with DTPMP-7Na according to OECD guideline 473 (Nakamura M, 2001) and in compliance with GLP, a dose related increase in the number of cells with aberrations was observed after 48-hour treatment in an in vitro chromosome aberration assay using Chinese hamster lung cells. No increase in aberrations was observed after 4 or 24-hour exposure or in the presence of metabolic activation. Appropriate positive and solvent controls were included and gave expected results. No evaluation of the effect of pH or of osmolality was made in the study. The 48-hour exposure time was included because test substance caused cell cycle delay. The study is not fully compliant with the current test guideline for in vitro cytogenicity: the number of cells per treatment was lower than the updated guideline; cytotoxicity was not evaluated by the criteria now required; the level of cytotoxicity observed at the top two concentrations in the 48 hour treatment was greater than that required by the guideline.

Mammalian mutagenicity

An aqueous solution of DTPMP-xNa, was tested in the mouse lymphoma L5178Y thymidine kinase assay using the microwell variant of the assay (Central Toxicology Laboratory, 1997). The maximum concentration tested was 2200 µg/ml. Higher concentrations were not used as the study authors considered that they gave excessively high osmolarity. A toxicity limit was not reached in these tests as the relative toxicity was >75%. No increases in mutation frequency were seen in any of these tests. Nevertheless, a higher dose level could have been employed in order to satisfy the criteria for a convincing assay.

In vivo chromosome aberration

While there is evidence for potential to induce chromosome aberrations in vitro, there is no evidence of clastogenicity in vivo. Evidence for a lack of genotoxic potential of DTPMP, and its salts, in vivo is provided by a well-conducted chromosome aberration study in rat bone marrow following gavage with doses up to 1970 mg DTPMP/kg bw (Hazleton Laboratories, 1983). The number of cells scored for aberrations and mitotic index was lower than that required in the current guideline. The top dose resulted in 25% mortality and loss of body weight. Metaphases were analysed from 4-6 animals per group. No evidence is presented in the study for the test substance reaching bone marrow. The mitotic index was unaffected by the administration of the test substance. However, there is sufficient evidence from other sources (Table 1) to provide information on exposure and toxicity to bone marrow, so in this respect the study does not stand alone. In the interests of animal welfare, use should be made of historical data and it is considered that in view of the clear negative result from the in vivo chromosome aberration assay, and a negative carcinogenicity study with the sodium salt (CAS 22042-96-2), it can be concluded that DTPMP (5-7Na) is not clastogenic.

The measurement of mitotic index might not be considered conclusive because an insufficient number of cells were scored. The mitotic index is a measure of toxicity, therefore the insufficient number of cells scored affects the measure of toxicity to bone marrow. There is sufficient evidence from other sources (Table 1) to provide information on exposure and toxicity to bone marrow, so in this respect the study does not stand alone. In the interests of animal welfare, use should be made of historical data and it is the lead registrant’s opinion that in view of the clear negative result from the in vivo chromosome aberration assay, this study should be considered sufficient to follow up the positive in vitro chromosome aberration result. The sample size for in vivo chromosome aberration tests was increased in order to increase the statistical power of the assay (OECD, 2015). The sample size in the study was 100 cells per animal with 6 animals per group, and therefore exceeded the size required when the guideline was updated in 1997 (100 cells per animal with 5 animals per dose), though it falls short of that required in the 2014 and 2016 OECD 475 guidelines. The positive in vitro result was observed in a study which was statistically less robust than a new study would be, because the in vitro chromosome aberration study does not comply with the updated guideline (OECD 473, 2016). In the interests of animal welfare and in view of:

- the lack of positive results in mammalian mutagenicity studies

- the deficiencies in the in vitro cytogenicity study,

- clear negative result from the in vivo chromosome aberration assay, the existing in vivo study is considered adequate to follow up the in vitro results.

Evidence for systemic exposure

Table 1 Toxicokinetic data for DTPMP 

Endpoint

Reference

Absorption and distribution of DTPMP following oral and dermal administration

Procter and Gamble, 1978

Distributionof DTPMP following intravenous administration

Subramanian et al, 1975

Distribution of DTPMP following intravenous administration

Goeckeler, 1987

Although there is no evidence from the in vivo chromosome aberration study that the substance reached the bone marrow, evidence of systemic exposure was reported in the in vivo chromosome aberration study in which test substance was administered at 200, 600 and 1970 mg/kg bw (Hazleton Laboratories, 1983). Clinical signs and three mortalities were recorded in the high dose group.

Though absorption of DTPMP has been shown to be low (approximately 2%) after oral dosing (Procter and Gamble, 1978), the data indicate that a small proportion of an oral dose is systemically available and this would primarily result in distribution to bone, with the remainder distributed to other tissues including the bone marrow and germ cells. Systemic availability following oral administration and affinity for bone was demonstrated following oral gavage with DTPMP, radiolabelled in the carbon atoms of the phosphonomethyl groups (Procter and Gamble, 1978). The proportion of radiolabel in the tissues was low as a result of the low absorption; the concentration in bone, (2.9 ± 0.79 μg/g) was nine times greater than any other tissue. Radioactivity was not detected in bone marrow. Very low levels were observed in blood (0.05 ± 0.05 μg/ g), plasma (0.03 ± 0.02 μg/g) and testes (0.05 ± 0.007 μg/g). The limit of detection of the method was not stated, however it is likely that the differences between bone marrow, blood and testes are not significant. Both bone marrow and testes have very good blood supply, so would be exposed to substances present in the blood.

Further information comes from evidence from published literature (Subramanian et al, 1975; Goeckeler, 1987), which demonstrates through the use of medical imaging in rats, that DTPMP (in the form of radiochemical complexes) is preferentially distributed to the outer bone rather than the bone marrow (following intravenous administration).

Intravenous administration of radiolabelled DTPMP salts to rabbits was followed by imagining and distribution analysis. DTPMP was excreted in the urine (70% at 4 hours), and DTPMP localised in the skeleton (Subramanian et al, 1975). At 4 hours after dosing with indium-113-labelled DTPMP, the ratio of % dose/1% body weight was as shown below. Whilst the radioassay analysis detects the complexed radionuclide, the observation of different distribution patterns and clearance rates when different phosphonate complexes of the same nuclide were compared in this study supports the authors’ conclusion that the properties of the phosphonate are important.

A similar study has been carried out using samarium-153 labelled DTPMP acid (Goeckeler, 1987). After intravenous administration to rats, the majority of the substance was eliminated in the urine, and much of the remainder had distributed to the bone (Table 2). Although the analysis detected the radiolabel and not directly detect the complexing agent, DTPMP-H, the stability of the chelation of the radionuclide by DTPMP means that the result adds to the evidence for distribution of DTPMP to the bone. The differences between the results of the two studies reflect the different time after intravenous administration that samples were taken. Taking into account the differences resulting from route of administration and sampling time, the studies show similarities between the distribution of radionuclide complexed DTPMP and that of carbon-labelled DTPMP. The results of the two radionuclide studies support the assumption of comparable exposure of bone marrow and germ cells.

Table 2 Distribution data for DTPMP

Tissue

Distribution

following oral

administration

(μg/kg) Procter and Gamble

(1978) (species: rat)

Distribution

following 2h

intravenous administration

(% dose/g) (2h)

Goeckeler (1987)

(species: rat)

Distribution

following 4h

intravenous

administration

(% dose/1% body

weight) Subramanian (1975)

(species: rabbit)

Distribution

following 4h

intravenous

administration

(% dose/1% body

weight) Subramanian (1975)

(species: rabbit)

Radiolabel

carbon

153-Sm

85-Sr

113m-In

Blood

0.05 ± 0.05

74

0.7

0.4

Plasma

0.03 ± 0.02

Not determined

Not determined

Not determined

Average bone

2.9 ± 0.79

30

8.1

4.8

Marrow

Below limit detection

Not determined

0.3

0.2

Muscle

Below limit detection

0.9

0.2

0.04

Kidney

0.17 ± 0.03

0.4

0.9

2.3

Liver

0.11 ± 0.02

0.3

0.3

0.2

Testes

0.05 ± 0.007

Not determined

Not determined

Not determined

The evaluation of genetic toxicity for REACH Registration is undertaken in order to make a decision on whether the registered substance should be classified for germ cell mutagenicity.

Taken together, the experimental data on absorption and distribution of DTPMP indicate that germ cells and bone marrow are exposed to similar very low levels of DTPMP. The exposure of workers and the general population would be expected to result in low levels of adsorption. The majority of the absorbed dose would be excreted via the kidneys, and the rest would distribute primarily to bone (Proctor and Gamble, 1978).

Although toxicity to bone marrow was not observed in the in vivo chromosome aberration study, systemic toxicity was seen indicating that DTPMP was available in the circulatory system. As the bone marrow has a very good blood supply bone marrow cells would have been exposed to levels that are relevant to germ cell mutagenicity. Therefore, it is considered that the lack of chromosome aberrations in the in vivo study are relevant to potential mutagenicity to germ cells.

Discussion of trends in the DTPMP category

Table 3 Summary of mutagenicity studies

Parameter

Results

Results

Reference and reliability

 

15827-60-8

salts

 

Bacterial mutagenicity

Negative

Negative

Acid: Monsanto 1981a (Reliability 1)

Salt:Japan Oilstuff Inspectors corporation, 2001a (Reliability 1)

Mammalian cytogenicity

No data

Positive

Salt:Japan Oilstuff Inspectors corporation, 2001b (Reliability 2)

Mammalian gene mutation (L5178Y)

Negative

Negative

Acid: SRI International, 1983; Microbiological Associates, 1983 (Reliabilities 2)

Salt: Central Toxicology Laboratory 1997 (Reliability 2)

Mammalian gene mutation (CHO/hprt)

Negative

 

Pharmakon Research 1984 (Reliability 2)

Chromosome aberrations in vivo

Negative

 

Monsanto, 1983 (Reliability 2)

Conclusions:

Neither the acid nor the sodium salt induce mutations in well-conducted studies in bacterial or mammalian cells.

The conclusion that DTPMP (5 -7Na) is not mutagenic in L5178Y cells is substantiated by consideration of:

(1) Lack of structural alerts for mutagenicity

(2) Lack of evidence for gene mutation potential in sub-mammalian systems and

(3) Lack of potential to induce gene mutations in a well-conducted assay investigating mutations at the hprt locus in CHO cells.

While there is some evidence for the induction of chromosome aberrations in vitro, there is no evidence of clastogenicity in vivo. Considering the clear negative result from the in vivo chromosome aberration assay, and a negative carcinogenicity study with the sodium salt (CAS 22042-96-2), it is concluded that DTPMP acid is not clastogenic. Consequently, DTPMP and its salts are not considered to pose a genotoxic hazard.

Although no studies have been conducted on other salts, their genotoxic properties are expected to be related to the phosphonic ion and therefore the results on the acid and sodium salts can be used to predict a lack of genotoxicity of the potassium and ammonium salts. Potassium ions are not expected to contribute to the genetic toxicity of the potassium salt: potassium is an abundant mineral and plays an important role in metabolism. Ammonium ions are not expected to contribute to genetic toxicity of the ammonia salt of DTPMP as there is no indication for genetic toxicity in the data available for the ammonia category (OECD, 2007a).

Justification for classification or non-classification

Based on the available data for DTPMP acid and sodium salts, no classification for genetic toxicity is required for DTPMP (5-7Na) according to Regulation (EC) No 1272/2008.