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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 19 March 2010 to 11 May 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conclusive, performed to a valid guideline (OECD TG 476, adopted 21 July 1997) and was conducted under GLP conditions. No deviations from the test methods were noted. The reliability has been assigned in accordance with 'practical guide 6: How to report read-across and categories' which states that the maximum reliability for a read-across study is 2. The study is considered to be adequate and reliable for the purpose of registration under REACH (Regulation (EC) No. 1907/2006) as part of a weight of evidence approach. Read-across in accordance with Annex XI, Section 1.5 of Regulation (EC) No. 1907/2006 (REACH) is justified on the following basis: Polyphosphoric acids, ammonium salt (also known as ammonium polyphosphate) is a mixture of oligomeric species of ammonium phosphate. When analysed the substance appears to consist mainly of ammonium orthophosphates, ammonium diphosphate and ammonium triphosphate. Phosphate is not considered to be mutagenic as it is frequently present within the buffers used for in vitro mutagenicity tests (i.e. as sodium or potassium phosphates). Therefore in order to assess the mutagenicity potential of ammonium polyphosphate it is the ammonium ion that is of interest. As such it is scientifically justified to read-across to other ammonium phosphates. Such as, ammonium dihydrogenorthophosphate and diammonium hydrogenorthophosphate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Polyphosphoric acids, ammonium salts
EC Number:
269-789-9
EC Name:
Polyphosphoric acids, ammonium salts
Cas Number:
68333-79-9
Molecular formula:
[NH4PO3]n
IUPAC Name:
Polyphosphoric acids, ammonium salts
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): ammonium dihydrogenorthophosphate
- CAS Number: 7722-76-1
- Physical state: white crystals
- Analytical purity: 99.8%
- Lot/batch No.: 0927299
- Expiration date of the lot/batch: 03 February 2011 (allocated by NOTOX, 1 year after receipt of the test substance)
- Storage condition of test material: at room temperature in the dark
- Stability under storage conditions: stable

Method

Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1, 3, 10, 33, 100, 333, 750, 1150 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (RPMI medium) treatment groups were used as the vehicle controls
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (RPMI medium) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Details on test system and experimental conditions:
Two mutagenicity experiments were performed. In the first experiment, L5178Y TK mouse lymphoma cells were treated with the test material at eight dose levels, in duplicate, together with vehicle and positive controls. The exposure groups used were as follows: 3 hour exposures both with and without metabolic activation (8% S9-mix).

In the second experiment, L5178Y TK mouse lymphoma cells were treated with the test material at eight dose levels, in duplicate, together with vehicle and positive controls. The exposure groups used were as follows: 3 hour exposure with metabolic activation (12% S9-mix), and 24 hours without metabolic activation.

For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period. During this culture period at least 4 x 10^6 cells (if possible) were subcultered every day in oder to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CEday2) and the mutation frequency (MF).

The dose range of test material was selected following the results of a preliminary toxicity test and was 1 to 1150 µg/mL. There was no evidence of any marked toxicity in the preliminary toxicity test up to and including the highest test substance concentration of 1150 µL.

The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Cell culture:

Horse serum was inactivated by incubation at 56 °C for at least 30 minutes.

Basic medium: RPMI 1640 Herpes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively), 1mM sodium pyruvate and 2mM L-glutamin.

Growth medium: Basic medium, supplemented with 10% (v/v) heat-activated horse serum (=R10 medium)

Exposure medium: For 3 hour exposure: Cells were exposed to the test substance in basic medium supplemented wuith 5% (v/v) heat-inactivated horse serum (R5-medium). For 24 hour exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium).

Selective medium: selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL triflurothymidine (TFT)

Non-selective medium: non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).

Environmental conditions:
All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80-100% (actual range 59-94%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0 °C (actual range 34.9 - 37.9 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 35.1 - 36.0 °C), humidity (with a maximum of 9%) and CO2 percentage (with a maximum of 1%) occured that were caused the opening and closing of the incubator door, but the time of these deviations did not exceed 1 hour. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Evaluation criteria:
Data evaluation and statistical procedures:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for biological relevance including a comparison of the results with the historical control data range.

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126 (ref. 12).

A test substance is considered positive (mutagenic) in the mutation assay if it induced a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reached a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:

Table 1 shows the cell counts of the cultures after 3 hours of treatment with various concentrations of ammonium dihydrogenorthophosphate and after 24 hours and 48 hours of subculture and the calculated suspension growth and the relative suspension growth.

Both in the absence and presence of S9 -mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 1150 µg/mL compared to the suspension growth of the solvent control.

Table 2 shows the cell counts of the cultures after 24 hours of treatment with various concentrations of ammonium dihydrogenorthophosphate and after 24 hours of subculture and the calculated suspension growth and relative suspension growth.

In the absence of S9 -mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 1150 µg/mL compared to the suspension growth of the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell lines
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

First mutagenicity test:

Table 3 shows the percentages of cell survival and the mutation frequencies for various concentrations of ammonium dihydrogenorthophosphate.

No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

No significant increase in the mutation frequency at the TK locus was observed after treatment with ammonium dihydrogenorthophosphate either in the absence or presence of S9-mix. The numbers of small and large colonies in the ammonium dihydrogenorthophosphate treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Second mutagenicity test:

Table 4 shows the percentages of cell survival and the mutation frequencies for various concentrations of ammonium dihydrogenorthophosphate.

No toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix.

No significant increase in the mutation frequency at the TK locus was observed after treatment with ammonium dihydrogenorthophosphate either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the ammonium dihydrogenorthophosphate treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

Table 1 – Dose range finding test; Cytotoxicity of ammonium dihydrogenorthophosphate (3 hours treatment)

dose (µg/mL)

cell count after 3 hours of treatment (cells/mL x 105)

cell count after 24 hours of subculture (cells/mL x 105)

cell count after 48 hours of subculture (cells/mL x 105)

SG1(x 105cells/mL)

RSG2(%)

without metabolic activation

SC

5.3

3.8

7.5

96

100

33

5.6

4.0

7.7

110

114

100

5.5

3.6

7.7

98

102

333

5.2

3.6

7.8

94

98

750

5.3

3.6

7.8

96

99

1150

5.1

3.4

7.9

87

90

with metabolic activation

SC

5.5

3.9

8.2

113

100

33

5.7

3.8

9.1

126

112

100

5.7

3.8

8.4

116

103

333

5.5

4.1

8.2

118

105

750

5.4

4.0

7.9

109

97

1150

5.3

3.6

8.7

107

95

 

 

Table 2 – Dose range finding test; Cytotoxicity of ammonium dihydrogenorthophosphate (24 hours treatment)

dose (µg/mL)

cell count after 3 hours of treatment (cells/mL x 105)

cell count after 24 hours of subculture (cells/mL x 105)

SG1(x 105cells/mL)

RSG2(%)

without metabolic activation

SC

5.7

8.1

37

100

33

6.1

8.1

40

107

100

6.4

7.5

38

104

333

5.9

7.7

37

100

750

4.9

7.5

29

80

1150

4.9

6.5

25.69

 

 

Note: all calculations were made without rounding off

SC = solvent control = RPMI medium

(1)   

(2)   

 

Table 3 – Dose range finding test: Cytotoxic and mutagenic response of ammonium dihydrogenorthophosphate in the mouse lymphoma L5178Y test system

dose (µg/mL)

RSG (%)

CEday2(%)

RSday2(%)

RTG (%)

Mutation frequency x 10-6

total

small

large

without metabolic activation: 3 hours treatment

 

 

SC1

 100

91

100

100

101

51

45

SC2

95

104

55

43

1

109

99

107

116

93

52

37

3

107

95

102

109

108

48

54

10

110

93

99

109

109

59

44

33

117

111

119

140

94

56

34

100

104

98

105

109

82

43

35

333

101

83

89

89

74

40

31

750

101

99

107

107

95

41

49

1150

93

102

110

102

84

47

34

MMS

67

54

58

39

1095

607

338

with 8% (v/v) metabolic activation: 3 hours treatment

 

 

SC1

100

97

100

100

106

54

46

SC2

97

83

38

41

1

112

105

109

122

82

45

34

3

112

93

96

107

94

41

49

10

99

99

103

102

88

55

29

33

99

94

97

96

85

49

33

100

100

101

104

104

90

51

35

333

95

98

101

96

94

58

32

750

88

97

100

88

97

53

39

1150

89

101

104

93

118

67

44

CP

73

48

49

36

1809

877

564

 

Table 4 – Experiment 2: Cytotoxic and mutagenic response of ammonium dihydrogenorthophosphate in the mouse lymphoma L5178Y test system

dose (µg/mL)

RSG (%)

CEday2(%)

RSday2(%)

RTG (%)

Mutation frequency x 10-6

total

small

large

without metabolic activation: 24 hours treatment

 

 

SC1

100

80

100

100

95

60

32

SC2

65

79

51

28

1

109

72

100

108

69

20

48

3

112

58

80

90

96

57

36

10

116

60

83

96

95

61

32

33

116

59

82

94

81

30

49

100

112

58

80

90

59

36

22

333

98

49

67

65

69

35

33

750

77

51

70

55

89

56

31

1150

58

45

61

35

134

60

70

MMS

73

37

51

37

1424

851

404

with 12% (v/v) metabolic activation: 3 hours treatment

 

 

SC1

100

65

100

100

88

53

33

SC2

72

89

46

40

1

94

58

85

80

120

74

42

3

95

120

174

165

38

21

17

10

91

60

88

79

78

37

39

33

97

58

84

81

110

66

41

100

88

48

69

61

160

110

44

333

90

97

140

127

85

54

28

750

90

60

88

79

115

65

46

1150

69

41

60

42

127

75

49

CP

15

33

48

7

935

668

212

 

Note: all calculations were made without rounding off

RSG = Relative suspension growth; CE = cloning efficiency; RS = Relative Survival; RTG = Relative Total Growth; SC = Solvent control = RPMI 1640 medium; MMS = methylmethanesulfonate; CP = cyclophosphamide

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Ammonium dihydrogenorthophosphate is not mutagenic in the TK mutation test system under the experimental conditions described in this report.
Executive summary:

The report describes the effects of ammonium dihydrogenorthophosphate on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in two independent experiments in the absence and presence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and β-napthaflavone).

In the first experiment, ammonium dihydrogenorthophosphate was tested up to concentrations of 1150 µg/mL (0.01 M) in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 hours. In the second experiment, ammonium dihydrogenorthophosphate was again tested up to concentrations of 1150 µg/mL, but in the absence and presence of 12% (v/v) S9-mix. The incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9-mix, respectively. No toxicity was observed at this dose level in the absence and presence of S9-mix.

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased 11-fold and 16-fold for MMS in the absenceof S9-mix, and by 19- and 11-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in theabsence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, ammonium dihydrogenorthophosphate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that ammonium dihydrogenorthophosphate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described.

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