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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

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:
Between 22 December 2009 and 05 February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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 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
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Principles of method if other than guideline:
Not applicable.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of GLP inspection: 15 September 2009 Date of Signature on GLP certificate: 26 November 2009
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Aluminium orthophosphate
EC Number:
232-056-9
EC Name:
Aluminium orthophosphate
Cas Number:
7784-30-7
Molecular formula:
Al.H3O4P
IUPAC Name:
[phosphato(3-)-kappa~3~O,O',O'']aluminum
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Aluminium orthophosphate
- Physical state: solid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): -
- Lot/batch No.: A88770A
- Expiration date of the lot/batch: 05 June 2010
- Stability under test conditions: Stable under normal temperatures and pressure
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
Histidine for Salmonella.
Tryptophan for E.Coli
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta­naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. The test material formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: at 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA100
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 1 µg/plate
Remarks:
With S9 mix
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: at 5µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1535
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
with S9 mix Migrated to IUCLID6: 2-Aminoanthracene at 2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: at 2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 10 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: at 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous muation rate for TA98
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix Migrated to IUCLID6: at 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rate for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: at 80 µg/plate
Untreated negative controls:
yes
Remarks:
Sponateous muation rate for TA1537
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene at 2 µg/plate
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 -72 hrs


SELECTION AGENT (mutation assays): Not applicable.


NUMBER OF REPLICATIONS: Triplicate plating.


NUMBER OF CELLS EVALUATED: Not applicable.


DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.


OTHER EXAMINATIONS: None
Evaluation criteria:
Acceptance Criteria:

The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10E9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Tested up to maximum recommended dose of 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material formed the best doseable suspension in dimethyl sulphoxide at 50 mg/ml in solubility checks performed in-house.
- Precipitation: A pale, fine precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-)

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

82

105

111

111

114

108

109

126

109

103

120P

+

TA100

104

94

99

50

63

83

69

82

90

77

92P

-

WP2uvrA-

26

26

20

29

26

32

15

25

19

29

28P

+

WP2uvrA-

28

32

30

25

31

26

29

27

28

24

25P

P         Precipitate

The test material caused a visible reduction in the growth of the bacterial background lawn of Salmonella strain TA100, without S9-mix, at 5000 µg/plate in the range-finding test only. However, this response was not observed in either the preliminary toxicity test or main test and was, therefore, considered spurious and of no biological relevance. No toxicity was noted to any of the remaining bacterial tester strains at any test material dose level in either the absence or presence of S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A pale, fine precipitate was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation for the Main test are presented in the tables below:

Spontaneous Mutation Rates (Concurrent Negative Controls)

Range-finding Test (Experiment 1)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

93

 

14

 

43

 

13

 

11

 

120

(110)

13

(16)

24

(33)

23

(19)

11

(12)

118

 

22

 

33

 

20

 

14

 

Main Test (Experiment 2)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

100

 

20

 

21

 

22

 

5

 

102

(101)

18

(19)

16

(20)

22

(22)

7

(7)

100

 

18

 

24

 

21

 

9

 

Test Results: Range-Finding Test– Without Metabolic Activation

Test Period

From: 17 January 2010

To: 20 January 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

102

95

99

(99)

3.5#

29

31

19

(26)

6.4

36

35

40

(37)

2.6

20

21

13

(18)

4.4

8

13

16

(12)

4.0

-

50

97

72

91

(87)

13.1

26

28

24

(26)

2.0

35

36

37

(36)

1.0

21

16

15

(17)

3.2

19

19

11

(16)

4.6

-

150

80

88

81

(83)

4.4

21

30

32

(28)

5.9

38

38

38

(38)

0.0

19

17

22

(19)

2.5

21

13

14

(16)

4.4

-

500

87

85

91

(88)

3.1

20

24

16

(20)

4.0

33

36

41

(37)

4.0

15

14

10

(13)

2.6

11

16

14

(14)

2.5

-

1500

91

94

90

(92)

2.1

24

21

16

(20)

4.0

37

36

41

(38)

2.6

22

11

20

(18)

5.9

11

15

13

(13)

2.0

-

5000

62 TP

64 TP

83 TP

(70)

11.6

21 P

30 P

12 P

(21)

9.0

31 P

43 P

34 P

(36)

6.2

18 P

21 P

28 P

(22)

5.1

14 P

16 P

14 P

(15)

1.2

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

530

657

665

(617)

75.7

1521

1622

1317

(1487)

155.4

913

956

898

(922)

30.1

153

156

188

(166)

19.4

595

896

1010

(834)

214.4

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

T        Partial absence of bacterial background lawn

#        Standard deviation

Test Results: Range-Finding Test– With Metabolic Activation

Test Period

From: 17 January 2010

To: 20 January 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

77

90

77

(81)

7.5#

18

16

25

(20)

4.7

46

38

47

(44)

4.9

24

29

21

(25)

4.0

19

17

16

(17)

1.5

+

50

90

63

71

(75)

13.9

9

9

12

(10)

1.7

34

41

49

(41)

7.5

21

25

27

(24)

3.1

10

20

12

(14)

5.3

+

150

97

77

C

(87)

14.1

17

20

26

(21)

4.6

38

48

45

(44)

5.1

30

24

21

(25)

4.6

9

15

17

(14)

4.2

+

500

85

68

77

(77)

8.5

18

29

21

(23)

5.7

30

40

41

(37)

6.1

17

27

21

(22)

5.0

15

15

13

(14)

1.2

+

1500

80

83

94

(86)

7.4

9

19

27

(18)

9.0

47

40

36

(41)

5.6

27

28

24

(26)

2.1

12

14

12

(13)

1.2

+

5000

75 P

77 P

76 P

(76)

1.0

22 P

22 P

21 P

(22)

0.6

37 P

47 P

39 P

(41)

5.3

22 P

16 P

27 P

(22)

5.5

16 P

5 P

11 P

(11)

5.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

846

1120

1065

(1010)

144.9

187

231

228

(215)

24.6

247

276

297

(273)

25.1

97

157

152

(135)

33.3

242

290

307

(280)

33.7


BP      Benzo(a)pyrene

2AA    2-Aminoanthracene

C        Contaminated

P        Precipitate

#        Standard deviation

Test Results: Main Test– Without Metabolic Activation

Test Period

From: 02 February 2010

To: 05 February 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

93

94

94

(94)

0.6#

13

19

18

(17)

3.2

18

17

17

(17)

0.6

20

19

19

(19)

0.6

15

8

8

(10)

4.0

-

15

89

98

98

(95)

5.2

19

18

16

(18)

1.5

19

19

16

(18)

1.7

15

15

16

(15)

0.6

13

15

13

(14)

1.2

-

50

79

80

81

(80)

1.0

22

22

16

(20)

3.5

16

15

15

(15)

0.6

24

15

22

(20)

4.7

12

15

10

(12)

2.5

-

150

83

87

87

(86)

2.3

19

19

20

(19)

0.6

16

16

15

(16)

0.6

22

18

18

(19)

2.3

12

14

12

(13)

1.2

-

500

84

86

84

(85)

1.2

17

15

17

(16)

1.2

20

18

18

(19)

1.2

15

20

20

(18)

2.9

12

12

11

(12)

0.6

-

1500

77

81

76

(78)

2.6

15

15

16

(15)

0.6

18

20

12

(17)

4.2

21

16

22

(20)

3.2

15

14

13

(14)

1.0

-

5000

98 P

100 P

97 P

(98)

1.5

22 P

17 P

15 P

(18)

3.6

19 P

15 P

22 P

(19)

3.5

22 P

22 P

16 P

(20)

3.5

13 P

14 P

9 P

(12)

2.6

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

552

593

439

(528)

79.8

436

460

470

(455)

17.5

645

681

685

(670)

22.0

152

154

149

(152)

2.5

735

691

703

(710)

22.7


ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation

FOR REST OF RESULTS SEE OVERALL REMARKS, ATTACHMENTS

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction. The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Methods. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with suspensions of the test material using both the Ames plate incorporation and pre-incubation methods at up to 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 for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test material formulations. The dose range was amended slightly following the results of the range-finding test and the change in test methodology and was 15 to 5000 μg/plate.

Results. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused a visible reduction in the growth of the bacterial background lawn of Salmonella strain TA100, without S9-mix, at 5000 μg/plate in the range-finding test only. However, this response was not observed in either the preliminary toxicity test or main test and was, therefore, considered spurious and of no biological relevance. No toxicity was noted to any of the remaining bacterial tester strains at any test material dose level in either the absence or presence of S9-mix. The test material was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate. A pale, fine precipitate was observed at 5000 μg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method

Conclusion. The test material was considered to be non-mutagenic under the conditions of this test.