Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial reverse mutation assay / Ames test): DTPMP-7Na was negative with and without activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (OECD Test Guideline 471 and in compliance with GLP) (Japan Oilstuff Inspectors Corporation, 2001a).
Cytogenicity in mammalian cells: DTPMP-7Na was positive in Chinese hamster lung CHL/IU cells (OECD Test Guideline and in compliance with GLP 473) (Japan Oilstuff Inspectors Corporation, 2001b).
Mutagenicity in mammalian cells: read-across from DTPMP-xNa; negative in mouse lymphoma L5278Y cells (similar to OECD Test Guideline 476 and in compliance with GLP) (Central Toxicology Laboratory, 1997).

Link to relevant study records

Referenceopen allclose all

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
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 aberration (%)

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 aberration (%)

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 valid in vitro mammalian chromosome aberration study in mammalian cell line CHL/IU with and without metabolic activation, 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, without metabolic activation. No dose-depended increase in the number of cells with aberrations was observed after 6 or 24 hours treatment, with and without metabolic activation. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP-7Na is positive for clastogenicity in vitro under the conditions of this 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
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 significant 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:
DTPMP-7Na has been tested for mutagenicity to bacteria in a valid gene mutation study in bacterial strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation, conducted according to OECD Test Guideline 473 and in compliance with GLP. No dose depended increase in the number of revertants was observed in any of the bacterial test strains when tested with or without metabolic activation up to cytotoxic concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP-7Na is negative for mutagenicity in bacteria under the conditions of this test.
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
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:
DTPMP-xNa has been tested for mutagenicity to mammalian cells in a valid gene mutation study in mouse lymphoma L5178Y cells with and without metabolic activation, conducted according to OECD Test Guideline 476 and in compliance with GLP. No increase in the mutant frequency was observed when tested with or without metabolic activation up to limit concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP-xNa is negative for mutagenicity in mouse lymphoma L5178Y cells under the conditions of this test.
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-H; negative (similar to OECD Test Guideline 475) (Hazleton Laboratories, 1983)

Link to relevant study records
Reference
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
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 increased.
- 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:
DTPMP-H has been tested for clastogenicity in a valid in vivo chromosome aberration study in rat bone marrow following gavage with doses up to 1970 mg DTPMP-H/kg bw (Hazleton Laboratories, 1983). The study was conducted according to a protocol similar to OECD Test Guideline 475 with deviations and in compliance with GLP. The deviations included lower number of cells scored for aberrations and mitotic index 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 was presented for the test substance reaching the bone marrow. In the study the mitotic index was unaffected by the administration of the test substance and there was 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
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Data are available for DTPMP-7Na from in vitro bacterial and mammalian cytogenicity studies. Data are available for DTPMP-xNa from an in vitro mammalian mutagenicity study. An in vivo chromosome aberration study in rats is available for DTPMP-H. See attachment to Section 13 for justification of read-across within DTPMP Category.

DTPMP-7Na has been tested for mutagenicity to bacteria in a valid gene mutation study in bacterial strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A with and without metabolic activation, conducted according to OECD Test Guideline 471 and in compliance with GLP. No dose-depended increase in the number of revertants was observed in any of the bacterial test strains when tested with or without metabolic activation up to cytotoxic concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP-7Na is negative for mutagenicity in bacteria under the conditions of this test (Japan Oilstuff Inspectors Corporation, 2001a).

DTPMP-7Na has been tested for clastogenicity in a valid in vitro mammalian chromosome aberration study in mammalian cell line CHL/IU with and without metabolic activation, 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, without metabolic activation. A not dose-dependent increase in the number of cells with aberrations was observed after 6 or 24 hours treatment, with and without metabolic activation. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP-7Na is positive for clastogenicity in vitro under the conditions of this test (Japan Oilstuff Inspectors Corporation, 2001b).

DTPMP-xNa has been tested for mutagenicity to mammalian cells in a valid gene mutation study in mouse lymphoma L5178Y cells with and without metabolic activation, conducted according to OECD Test Guideline 476 and in compliance with GLP. No increase in the mutant frequency was observed when tested with or without metabolic activation up to limit concentration. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that DTPMP-xNa is negative for mutagenicity in mouse lymphoma L5178Y cells under the conditions of this test (Central Toxicology Laboratory, 1997).

DTPMP-H has been tested for clastogenicity in a valid in vivo chromosome aberration study in rat bone marrow following gavage with doses up to 1970 mg DTPMP-H/kg bw (Hazleton Laboratories, 1983). The study was conducted according to a protocol similar to OECD Test Guideline 475 with deviations and in compliance with GLP. The deviations included lower number of cells scored for aberrations and mitotic index 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 was presented for the test substance reaching the bone marrow. In the study the mitotic index was unaffected by the administration of the test substance and there was 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.

In line with ECHA Final Decision number CCH-D-2114495836-29-01/F an in vivo mammalian alkaline comet assay in rats (oral route; tissues: liver, glandular stomach and duodenum), according to OECD Test Guideline 489, has been planned for the Category member DTPMP (5-7Na) in order to investigate the positive effects seen in vitro.

Justification for classification or non-classification

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