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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 May 1995 to 13 June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (is a mixture of ammonium orthophosphates and ammonium pyrophosphates and is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates and pyrophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate or pyrophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus, the target substance (diammonium dihydrogenpyrophosphate) and the source substance (mixture of ammonium orthophosphates and pyrophosphates) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the toxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection: 31 January 1994 Date of signature on certificate: 16 March 1994
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Ammonium polyphosphates impurities not otherwise specified
Molecular formula:
not applicable
IUPAC Name:
Ammonium polyphosphates impurities not otherwise specified
Test material form:
liquid
Details on test material:
The identity of the test material is not reported within the study report itself, however the data is referred to in the Toxicological Risks of Selected Flame Retardant Chemicals (2000), Subcommittee on Flame-Retardant Chemicals, Committee on Toxicology, Board on Environmental Studies and Toxicology, National Research Council. ISBN: 0-309-59232-1. The substance LR-2 is an ‘ammonium polyphosphate’ and the author provides the following additional information with regards to the chemical identity of LR2: ‘Based on information provided by the manufacturer (Stewart Miller, Albright and Wilson, pers. commun., Nov. 1, 1999), a typical species distribution of polyphosphates in LR2 is 20% orthophosphate, 40% pyrophosphate,
Specific details on test material used for the study:
The identity of the test material is not reported within the study report itself, however the data is referred to in the Toxicological Risks of Selected Flame Retardant Chemicals (2000), Subcommittee on Flame-Retardant Chemicals, Committee on Toxicology, Board on Environmental Studies and Toxicology, National Research Council. ISBN: 0-309-59232-1. The substance LR-2 is an ‘ammonium polyphosphate’ and the author provides the following additional information with regards to the chemical identity of LR2: ‘Based on information provided by the manufacturer (Stewart Miller, Albright and Wilson, pers. commun., Nov. 1, 1999), a typical species distribution of polyphosphates in LR2 is 20% orthophosphate, 40% pyrophosphate,

Method

Target gene:
Histidine for Salmonella.
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA100, TA1537, TA1538, TA98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0, 25, 75, 250, 750, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 4-nitro-o-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration: approximately 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: evaluation of number of revertant colonies and examined for a thinning of the background lawn
Evaluation criteria:
For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results or equivocal results are obtained, then a third experiment may be used to confirm the correct response. All data are statistically analysed using the methods recommended by UKEMS (5) and normally Dunnett's method of linear regression is used to evaluate the result. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity, solubility or up to the maximum recommended dose of 5000 µg/plate. In this case the limiting factor was the maximum recommended dose.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 100, TA 1535, TA1538, TA98, TA1537
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Study

The dose range of the test material used in the preliminary toxicity study was, (after a purity allowance), 0, 25, 75, 250, 750 and 2500 µg/plate. The maximum dose level was not achieved in the preliminary toxicity test, this is not considered significant as there was no toxicity noted in the main experiments at any dose level tested. The test material was non-toxic in the strain of Salmonella used (TA 100).

The mean numbers of revertant colonies for the toxicity assay were:

Strain

Dose (µg/plate)

0

25

75

250

750

2500

TA100

110

107

100

123

117

109

Mutation Study

The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were aU found to be satisfactory.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls both with and without metabolic activation are presented in Tables 2 and 3 for experiment 1 and Tables 4 and 5 for experiment 2.

Key to Table of Test Results

1 . "Number of revertants" - The observed values and average value (in parentheses) are shown in order, beginning with the lowest concentration of the test substance.

2. Figures immediately below average values refer to standard deviation.

3 . The following postfixes are used when required:

C = contaminated

P = precipitate

S =sparse background lawn

v = very thin background lawn

T = toxic

x = plate unscorable

Table 1: Spontaneous Mutation Rates

Experiment 1:

Number of Revertants (Number of Colonies per plate)

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA1538

TA98

TA1537

112

 

20

 

23

 

19

 

20

 

100

(98)

27

(23)

24

(22)

14

(17)

19

(17)

82

 

23

 

20

 

18

 

12

 

 

Experiment 2:

Number of Revertants (Number of Colonies per plate)

Base-pair Substitution Type

Frameshift Type

TA100

TA1535

TA1538

TA98

TA1537

116

 

22

 

15

 

22

 

12

 

105

(102)

17

(19)

13

(22)

27

(17)

12

(10)

85

 

19

 

20

 

17

 

5

 

Table 2:

Experiment 1 – Without Metabolic Activation

With or without S9-mix

Test substance concentration µg/plate

Number of revertants (Number of colonies per plate)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

TA 1538

TA 98

TA 1537

 

-

 

0

153

 

25

 

15

 

22

 

17

 

164

(149)

17

(22)

17

(18)

24

(21)

12

(12)

131

16.8

24

4.4

23

4.2

18

3.1

8

4.5

 

-

 

25

144

 

17

 

19

 

17

 

9

 

139

(137)

15

(16)

20

(16)

25

(23)

12

(8)

129

7.6

15

1.2

10

5.5

28

5.7

4

4.0

 

-

 

75

136

 

13

 

23

 

23

 

14

 

122

(125)

18

(14)

19

(18)

19

(21)

12

(12)

118

9.5

12

3.2

12

5.6

22

2.1

9

2.5

 

-

 

250

137

 

17

 

27

 

27

 

9

 

123

(127)

10

(15)

25

(19)

25

(23)

12

(9)

121

8.7

18

4.4

17

1.0

17

5.3

5

3.5

 

-

 

750

125

 

19

 

14

 

14

 

9

 

118

(132)

17

(19)

19

(18)

19

(18)

12

(10)

154

19.1

20

1.5

20

4.0

20

3.2

8

2.5

 

-

 

2500

136

 

13

 

32

 

32

 

8

 

127

(128)

15

(14)

27

(19)

27

(26)

10

(10)

120

8.0

15

1.2

19

4.0

19

6.6

13

2.5

 

-

 

5000

90

 

10

 

19

 

19

 

8

 

85

(80)

10

(9)

15

(21)

15

(19)

3

(6)

64

14.0

8

1.2

22

4.9

22

3.5

8

2.9

Positive controls

Name

ENNG

ENNG

4NOPD

4NOD

9AA

Concentration (µg/plate)

3

5

5

0.2

80

 

S9-mix

-

Number of colonies per plate

869

 

312

 

296

 

172

 

447

 

802

(794)

334

(308)

249

(267)

184

(182)

495

(444)

712

79.8

277

28.7

255

25.5

189

8.7

391

52.1

ENNG = N-ethyl-N’-nitro-N-nitrosguanidine 4NOPD = 4-nitro-o-phenylenediamine

4NQO = 4-nitroquinoline-1-oxide 9AA = 9-aminoacridine

Table 3:

Experiment 1 – With Metabolic Activation

With or without S9-mix

Test substance concentration µg/plate

Number of revertants (Number of colonies per plate)

Base-pair substitution type

Frameshift type

TA 100

TA 1535

TA 1538

TA 98

TA 1537

 

+

 

0

105

 

25

 

33

 

25

 

18

 

87

(98)

14

(16)

45

(32)

33

(31)

25

(24)

103

9.9

10

47.8

17

14.0

34

4.9

28

5.1

 

+

 

25

106

 

17

 

33

 

41

 

23

 

97

(100)

10

(15)

30

(27)

34

(36)

20

(24)

97

5.2

19

4.7

17

8.5

32

4.7

28

4.0

 

+

 

75

105

 

25

 

30

 

35

 

22

 

115

(109)

17

(19)

22

(25)

18

(29)

23

(23)

105

5.5

15

5.3

19

5.7

33

9.3

25

1.5

 

+

 

250

111

 

19

 

25

 

33

 

20

 

96

(103)

13

(15)

27

(26)

28

(28)

20

(21)

101

7.6

12

3.8

27

1.2

22

5.5

24

2.3

 

+

 

750

96

 

13

 

32

 

19

 

14

 

111

(103)

24

(19)

24

(29)

32

(27)

18

(15)

101

7.6

19

5.5

32

4.6

29

6.8

14

2.3

 

+

 

2500

107

 

14

 

27

 

19

 

17

 

116

(111)

10

(12)

36

(30)

34

(31)

23

(18)

111

4.5

12

2.0

27

5.2

40

10.8

15

4.2

 

+

 

5000

105

 

12

 

29

 

28

 

12

 

102

(97)

8

(10)

32

(28)

20

(27)

12

(14)

84

11.4

9

2.1

22

5.1

33

6.6

17

2.9

Positive controls

Name

2AA

2AA

2AA

2AA

2AA

Concentration (µg/plate)

1

2

0.5

0.5

2

 

S9-mix

+

Number of colonies per plate

732

 

110

 

294

 

276

 

219

 

803

(857)

139

(129)

191

(252)

220

(241)

232

(229)

1035

158.5

138

16.5

271

54.1

227

30.5

229

6.8

2AA = 2-aminoanthracene

For Table 4 and 5 - Experiment 2, please see attached document

Applicant's summary and conclusion

Conclusions:
The test material was found to be non-mutagenic under the conditions of the test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA99 and TA100 were treated with the test material using the Ames plate incorporation method at six dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was, (after a purity allowance of approximately 50%), 25 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as experiment 1, fresh cultures of the bacterial strains and fresh chemical formulations. The method used conforms with the OECD Guidelines for the Testing of Chemicals, Protocol No. 471 and also with Method B 14 in Commission Directive 92/69/EEC.

The vehicle (sterile distilled water) control plates produced counts of revertant colonies within the normal range.

All of the positive control chemicals used in the test produced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

The test material caused no visible reduction in the growth of the bacterial lawn at any of the dose levels in any of the strains of Salmonella tested. The test material was therefore, tested up to the maximum recommended dose of 5000 µg/plate.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation. The test material was found to be non-mutagenic under the conditions of this test