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) (Italmatch, 2003)
Cytogenicity in mammalian cells: read-across from DTPMP-7Na (CAS number 22042-96-2); positive in Chinese hamster lung IU cells (OECD TG 473) (Japan Oilstuff Inspectors Corporation, 2001b)
Mutagenicity in mammalian cells: negative in Chinese hamster ovary cells (similar to OECD TG 476) (Pharmakon Research International, 1984)

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

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

NUMBER OF REPLICATIONS: duplicate plates

DETERMINATION OF CYTOTOXICITY
- Method: other: growth inhibition
Evaluation criteria:
"If the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was judged positive."
Statistics:
Not done
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=313 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>=625 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: test substance supplied as a solution in water
- Precipitation: no precipitate seen
- Other confounding effects: no data

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

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

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

Concentration (µg/plate)

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0

134

128

23

14

19

27

18

39

16

23

1.2

137

131

21

13

20

30

19

35

16

26

4.9

127

142

18

16

24

26

16

33

17

22

20

114

127

28

16

20

34

18

32

19

17

78

132

130

18

13

29

30

18

31

14

22

313

46*

114

8*

8

21

18

11*

20

5*

12

1250

0*

125*

0*

4*

0*

17*

0*

10*

0*

4*

5000

0*

0*

0*

0*

0*

0*

0*

0*

0*

0*

Positive control

486

842

461

344

135

595

511

226

1767

82

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

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

Concentration (µg/plate)

TA 100

TA 1535

WP2 uvrA

TA 98

TA 1537

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0

117

137

17

13

27

26

17

43

8

22

10

137

NT

21

NT

NT

NT

15

NT

9

NT

20

124

NT

15

NT

NT

NT

21

NT

11

NT

39

124

125

9

11

22

27

22

37

9

23

78

125

136

15

10

27

31

11

37

12

15

156

116

133

16

17

20

26

17

39

6*

13

313

50*

115

6*

9

16

19

11*

32

6*

12

625

NT

106*

NT

4*

10*

21

NT

23*

NT

5*

1250

NT

108*

NT

2*

0*

13*

NT

3*

NT

5*

Positive control

496

810

451

321

160

563

588

200

1739

69

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

Conclusions:
In a reliable study, conducted to Japanese guidelines on mutagenicity tests (notification nos. 77 and 653), no genotoxicity was seen in a bacterial mutagenicity assay at up to 1250 µg/plate. The study was performed in compliance with Japanese guideline notification no. 76.
Endpoint:
in vitro 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 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:
31-Aug-1983 to 08-Feb-1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
inadequacies with respect to concentrations of submission substance tested
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
(some omissions from study report with respect to test system itself)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: F12
- 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:
other: CHO-K1-BH4
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 preparation at 2%
Test concentrations with justification for top dose:
3000, 5000, 6000, 7000, 8000 µg Dequest 2060/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: none (test substance diluted in F12 culture medium)
Untreated negative controls:
yes
Remarks:
F12 culture medium only
Negative solvent / vehicle controls:
no
Remarks:
test substance diluted in F12 culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
200 µg/ml without metabolic activation
Untreated negative controls:
yes
Remarks:
F12 culture medium only
Negative solvent / vehicle controls:
no
Remarks:
test substance diluted in F12 culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
100 µg/ml with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 19 hours
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
To be considered positive if:
a) The mean mutation frequencies of at least one of the 3 highest test article concentrations, with a mean survival rate of at least 10% are significantly
greater than that of the solvent control (p<=0.01 based on pooled inter-group variance); and,
b) The change in mean mutation frequency with increasing test article concentration exhibits a significant (p<=0.01) linear component of the dose-response relationship up to a maximum toxicity level of 90%."
To be considered negative if:
a) None of the mean mutation frequencies of any of the 3 highest test article concentrations with a mean survival rate of at least 10% are significantly
greater than that of the solvent control (p<=0.01) and,
b) The change in mean mutation frequency with increasing test article concentration does not exhibit a statistically significant (p<=0.01) linear
component of the dose-response relationship up to a maximum toxicity level of 90%.
Statistics:
Statistical analysis: Students t-test for group mean comparison and ANOVA on transformed data (performed on second test only).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Relative survival at 8000 µg/ml: -S9 65%, +S9 44%
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no data

RANGE-FINDING/SCREENING STUDIES: Preliminary test to establish optimum level of S9 preparation

COMPARISON WITH HISTORICAL CONTROL DATA: no data

ADDITIONAL INFORMATION ON CYTOTOXICITY: Two preliminary cytotoxicity tests were performed
1. no cytotoxicity at up to 1000 µg/ml with up to 10% S9 preparation
2. 84% survival at 6000 µg/ml in the absence of S9; 58, 62, 44 and 52% survival at 6000 µg/ml in the presence of 1, 2, 5 and 10% S9 preparation

Top dose is higher than the value of 5000 µg/ml normally considered the maximum for this test.

Note however this may represent the test solution as supplied, rather than the active acid.

Table 1 Preliminary mutagenicity assay - Dequest 2060 1% activation

Concentration µg/ml

+/-S9

Post treatment survival

Number of replicates

Mutation frequency (Mutants/10x6)

Solvent Control

-

97

2

 

1.7

-

102

1

+

98

1

 

3.6

+

97

6

2500

-

101

2

 

1.2

-

99

0

6000

-

76

2

 

2.4

-

72

2

7500

-

26

15

 

15.1

-

24

14

2500

+

88

0

 

1.2

+

85

2

6000

+

71

2

 

2.6

+

49

3

7500

+

40

5

 

3.9

+

39

2

Positive control

-

60

193

 

325.2

-

57

183

 

Table 2 Preliminary mutagenicity assay - Dequest 2060 2%, 5%, 10% activation

Concentration µg/ml

+/-S9

Post treatment survival

Number of replicates

Mutation frequency (Mutants/10x6)

Solvent Control

-

97

2

 

1.7

-

102

1

+

100

5

 

6.1

+

95

6

2500

2% +

85

0

 

1.9

2% +

92

3

6000

2% +

74

4

 

4.9

2% +

76

5

7500

2% +

36

12

 

10.2

2% +

38

7

Positive control

2% +

19

126

 

298.4

2% +

22

108

Solvent Control

+

107

7

 

4.7

+

87

1

2500

5%+

77

2

 

2.6

5%+

81

3

6000

5%+

72

4

 

6

5%+

76

7

7500

5%+

33

6

 

9

5%+

35

9

Solvent Control

+

102

1

 

2.6

+

91

4

2500

10%+

100

0

 

0

10%+

94

0

6000

10%+

72

7

 

9.5

10%+

63

8

7500

10%+

42

6

 

6.4

10%+

42

5

 

Table 3 Mammalian cell forward gene mutation assay

Concentration µg/ml

+/-S9

Post treatment survival

Number of replicates

Mutation frequency (Mutants/10x6)

Solvent Control

-

100.6.

1

0.4

-

99.4

0

-

97.2

0

3000

-

95

0

0

-

107.2

0

-

99.4

0

5000

-

98.8

0

0

-

103.5

0

-

87.2

0

6000

-

97.8

3

1.8

-

99.3

0

-

103.2

1

7000

-

91.9

0

2.5

-

71.2

6

-

82.8

1

8000

-

59.6

0

0.5

-

70.3

1

-

65.9

0

Positive control

-

69

129

310.3

-

58.4

222

-

57.3

216

Control

+

103.2

0

2.6

+

102.1

4

+

93.2

2

3000

+

79.4

1

0.6

+

65.1

0

+

97.5

0

5000

 

+

86.4

0

6.4

+

84.5

12

+

62.8

2

6000

+

80.5

12

6.5

+

66.8

3

+

71.1

0

7000

+

48.9

0

6.5

+

54.3

14

+

52.6

1

8000

+

53.2

2

2.9

+

38.4

0

+

43.7

5

Positive control

+

17.1

136

345

+

21

109

+

25.4

121

 

Conclusions:
In a reliable study, conducted using a protocol similar to the OECD guideline 476, no evidence of mutagenic potential was detected in Chinese hamster ovary cells in vitro in the absence or presence of an exogenous metabolic activation system in an assay evaluating mutation at the HGPRT locus. The study was performed in compliance with GLP.
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): Negative (similar to OECD TG 475)

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 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Information is available for DTPMP-H from bacterial and mammalian mutagenicity studies, and from an in vivo chromosome aberration study in rats. Information is available from in vitro cytogenicity study on the related substance, DTPMP-7Na. The use of read-across data between members of the category is in accordance with the rationale outlined in the read-across justification included in Section 13.

Bacterial mutagenicity

The results of all the bacterial mutagenicity studies were negative.

In the key study, conducted to Japanese guidelines on mutagenicity tests (notification nos. 77 and 653), no genotoxicity was seen in a bacterial mutagenicity assay for DTPMP-H at concentrations up to 1250 µg/plate when tested with or without metabolic activation in Salmonella typhimurium TA98, TA100, TA 1535, TA 1538 and E. coli WP2 uvrA (Italmatch, 2003).

The mutagenic potential of a 50% aqueous solution of DTPMP-H was investigated in a well conducted standard guideline test using the plate incorporation method with strains TA98, TA100, TA1535 and TA 1537 (Monsanto, 1981). The top doses used were 10 µl/plate with S9 mix and 0.3 µl/plate without S9 mix and toxicity was induced at the highest doses, consistent with the high toxicity of phosphonic acids in this test system. No statistically significant increase in mutant numbers was seen at any concentration. This negative result is confirmed with the results of a briefly reported study with strains TA98, 100, 1535, 1537 and 1538 with the same test substance (Monsanto, 1977).

In a recent well-conducted assay an aqueous solution containing DTPMP-7Na (pH 6-8) was tested for the induction of gene mutations in Salmonella typhimurium strains TA98, 100, 1537 and 1535 and in Escherichia coli strain WP2 uvr- (Japan Oilstuff Inspectors Corporation, 2001a). The study was conducted using the preincubation modification in the presence and absence of S9 mix on two occasions. The test substance did not induce mutations in either strain at concentrations up to 5000 µg/plate, the accepted upper limit for this assay.

In vitro cytogenicity

In the key cytogenicity assay, which was conducted to the appropriate Japanese guideline an aqueous solution containing DTPMP-7Na was tested for the induction of chromosomal aberrations in CHL IU cells. (Japan Oilstuff Inspectors Corporation, 2001b). A delay in the cell cycle was observed in the continuous test with 24 hours treatment, so a 48 hour treatment (3 cell cycles) was carried out. The 4 hour treatment was conducted in the presence and absence of phenobarbitone and 5,6-benzoflavone induced S9 mix. A dose-related increase in the number of structural aberrations was observed in the 48 hour treatment group. It was concluded that the test substance has a clastogenic potential in the CHL/IU cell line. 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

Results are available from four mammalian mutagenicity studies on DTPMP-H. In some of the studies the test substance was neutralised.

In the oldest study, DTPMP-H was tested in a mammalian mutagenicity study conducted according to a protocol similar to the OECD Test Guideline 476 and in compliance with GLP (SRI International, 1982). The authors of the report concluded that mutagenicity was seen in mouse lymphoma L5178Y cells tested up to the limit of toxicity in both the presence and absence of S9. However, in the absence of metabolic activation the toxicity in one of the dose levels reported to be positive was too great to be acceptable under the current guideline, and the increase in mutant frequency at the dose with acceptable toxicity was less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria. In the presence of metabolic activation, the positive result in the first two experiments was probably caused by reduction in pH. The study report referred to a follow up study using neutralised test solution and it was concluded by the study authors that the results were not due to pH but there is insufficient detail provided to assess whether pH was adequately controlled. Since the pH was likely to have been low at high concentrations the result is considered to be unreliable and the study is disregarded.

A second study is available which was conducted according to a protocol similar to OECD Test Guideline 476 and in compliance with GLP in order to evaluate the possiblity that the mutagenicity reported in an earlier study was not an artefact of pH (SRI International, 1983). The neutralised test substance was tested in mouse lymphoma L5178Y cells tested to the limit of toxicity in the presence of metabolic activation. The study authors reported that the result was positive in the presence of metabolic activation. However, the increases in mutant frequency were less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria.

The third study in mouse lymphoma L5178Y cells was conducted according to a protocol similar to OECD Test Guideline 476 (1997) and in compliance with GLP, mutagenicity was reported when tested to the limit of toxicity in the presence metabolic activation (Microbiological Associates, 1983). The study was conducted in compliance with GLP. However, the increases in mutant frequency were less than the control value plus the global evaluation frequency for the plate method, so would not be considered positive by currently accepted criteria.  

The potential for DTPMP-H to induce gene mutations in mammalian cells has also been investigated in CHO cells using forward mutation at the hprt locus (Pharmakon Research International, 1984). This well-conducted study exceeded OECD Test Guidelines. The top dose (8000 µg/ml) exceeded that normally considered the maximum for this assay and resulted in moderate cytotoxicity (relative survival 65% and 44% in presence and absence of S9, respectively). However, it was not clear whether the test solution, or active acid was tested. No evidence of mutagenic potential was seen at the hprt locus in CHO cells in the absence or presence of S9 at four different levels.

An aqueous solution of DTPMP-xNa (CAS 22042-96-2), neutralized with NaOH, 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.

Osmolarity was not assessed in the in vitro studies on the acid; it is an unlikely cause of the positive response because positive responses are only seen consistently in the presence of metabolic activation. These substances are strong chelators and there is some unpublished evidence that other chelators may produce false positive responses in this assay, presumably due to the removal of essential metal ions such as Zn, Mg etc. Genetic toxicity studies conducted with the structurally-analogous phosphonate ATMP are available. It has been observed that DTPMP acid produces a positive response in this assay yet ATMP does not (SIAR, 2005), which is consistent with their relative chelation potentials. Thus there is considerable evidence that the positive response with the acid is an isolated and artefactual response resulting from pH. The elevated mutant frequency with neutralised acid is below the frequency at which the study would be considered to be positive according to currently accepted criteria.

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-H, 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-H/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-H 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-xNa 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 reliable in vitro and in vivo data, DTPMP-H does not require classification according to Regulation (EC) No 1272/2008.