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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 26 May 2010 and 24 June 2010.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conclusive, done to a valid guideline (OECD TG 471) and was conducted under GLP conditions. No deviations from the test methods were noted.

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:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Orthophosphoric acid
EC Number:
231-633-2
EC Name:
Orthophosphoric acid
Cas Number:
7664-38-2
Molecular formula:
H3O4P
IUPAC Name:
phosphoric acid
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): PHOSPHORIC ACID TECHNICAL GRADE
- Analytical purity: 85.01%
- Lot/batch No.: HU10009
- Expiration date of the lot/batch: Not supplied
- Storage condition of test material: Room temperature in the dark
- Other: Description: Clear colourless slightly viscous liquid

Method

Target gene:
Histidine operon for Salmonella
Tryptophan operon 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: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully miscible in sterile distilled water at 50 mg/ml in solubility checks performed in house. Sterile distilled water was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 1 µg/plate
Remarks:
TA100 strain. Used with metabolic activation (S9 mix) only
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
TA1535 strain. Used with metabolic activation (S9 mix) only
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 2 µg/plate
Remarks:
TA1535 strain. Used with metabolic activation (S9 mix) only.
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA-
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene: 10 µg/plate
Remarks:
WP2uvrA- strain. Used with metabolic activation (S9 mix) only
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA98
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1537
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA100
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water.
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of TA1535
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates of WP2uvrA
Negative solvent / vehicle controls:
yes
Remarks:
Sterile distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours at 37°C - all plates.
- Pre-incubation period: Only experiment 2 contained a pre-incubation period.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: the frequency of revertant colonies assessed using a Domino colony counter.

The positive and untreated controls were dosed using the standard plate incorporation method described in Section "Mutation Test - Experiment 1".


Preliminary Toxicity Test
In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate). Ten concentrations of the test material formulation and a vehicle control (sterile distilled water) were tested. In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37°C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Method: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.

Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 µg/plate) but the formulations were pre-incubated.
Method: Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.1 ml of the vehicle or test material formulation and incubated for 20 minutes at 37°C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.


All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.




Evaluation criteria:
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 (6) 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
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:
RESULTS
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.
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 88 80 111 93 98 86 90 93 76 99 74
+ TA100 98 74 102 88 109 92 80 114 97 90 99
- WP2uvrA- 22 27 18 23 24 19 22 25 21 22 22
+ WP2uvrA- 19 32 30 31 22 23 25 24 22 20 23

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9‑mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable (not presented here).

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 Table 1 and Table 2 for Experiment 1 and Table 3 and Table 4 for Experiment 2.
A history profile of vehicle and positive control values for 2008 and 2009 is presented in Appendix 2 (attached).
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates of any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, 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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test Results: Experiment 1 – Without Metabolic Activation

 

Test Period

From: 08 June 2010

To: 11 June 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

108

92

89

(96)

10.2#

22

29

25

(25)

3.5

23

32

27

(27)

4.5

22

15

27

(21)

6.0

16

14

10

(13)

3.1

-

50

86

99

115

(100)

14.5

26

27

27

(27)

0.6

33

29

27

(30)

3.1

14

21

14

(16)

4.0

10

15

16

(14)

3.2

-

150

107

111

97

(105)

7.2

20

25

24

(23)

2.6

24

30

24

(26)

3.5

20

18

15

(18)

2.5

15

16

13

(15)

1.5

-

500

98

103

102

(101)

2.6

29

21

23

(24)

4.2

32

26

30

(29)

3.1

20

19

18

(19)

1.0

12

7

15

(11)

4.0

-

1500

103

103

117

(108)

8.1

23

20

18

(20)

2.5

27

31

26

(28)

2.6

16

14

22

(17)

4.2

10

15

10

(12)

2.9

-

5000

111

119

112

(114)

4.4

25

19

22

(22)

3.0

29

27

22

(26)

3.6

13

21

19

(18)

4.2

12

13

11

(12)

1.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

499

514

504

(506)

7.6

306

269

231

(269)

37.5

208

189

240

(212)

25.8

145

145

152

(147)

4.0

559

652

385

(532)

135.5

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

 

Table 2. Test Results: Experiment 1 – With Metabolic Activation

 

Test Period

From: 08 June 2010

To: 11 June 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

103

103

87

(98)

9.2#

14

14

13

(14)

0.6

26

30

36

(31)

5.0

24

25

31

(27)

3.8

9

14

14

(12)

2.9

+

50

104

110

99

(104)

5.5

13

7

10

(10)

3.0

25

30

31

(29)

3.2

21

18

23

(21)

2.5

10

15

14

(13)

2.6

+

150

119

100

93

(104)

13.5

12

13

14

(13)

1.0

30

34

32

(32)

2.0

23

25

25

(24)

1.2

13

10

7

(10)

3.0

+

500

90

84

92

(89)

4.2

14

14

15

(14)

0.6

30

24

26

(27)

3.1

18

24

29

(24)

5.5

15

11

11

(12)

2.3

+

1500

104

107

100

(104)

3.5

12

9

14

(12)

2.5

35

29

29

(31)

3.5

27

23

33

(28)

5.0

10

15

9

(11)

3.2

+

5000

86

98

84

(89)

7.6

14

14

12

(13)

1.2

30

30

27

(29)

1.7

24

23

32

(26)

4.9

12

9

14

(12)

2.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

405

389

381

(392)

12.2

219

197

229

(215)

16.4

300

323

324

(316)

13.6

202

133

128

(154)

41.4

242

273

216

(244)

28.5

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

 

 

 

Table 3. Test Results: Experiment 2 – Without Metabolic Activation

 

Test Period

From: 21 June 2010

To: 24 June 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

122

107

101

(110)

10.8#

18

9

15

(14)

4.6

30

21

33

(28)

6.2

32

26

20

(26)

6.0

20

19

15

(18)

2.6

-

50

98

122

98

(106)

13.9

15

11

18

(15)

3.5

35

24

42

(34)

9.1

18

20

23

(20)

2.5

9

27

8

(15)

10.7

-

150

84

101

110

(98)

13.2

13

15

8

(12)

3.6

33

25

31

(30)

4.2

27

16

24

(22)

5.7

12

14

18

(15)

3.1

-

500

123

113

122

(119)

5.5

12

8

13

(11)

2.6

25

43

25

(31)

10.4

26

24

35

(28)

5.9

15

10

16

(14)

3.2

-

1500

112

106

97

(105)

7.5

11

13

15

(13)

2.0

33

37

33

(34)

2.3

31

20

23

(25)

5.7

11

14

14

(13)

1.7

-

5000

89

100

101

(97)

6.7

7

8

20

(12)

7.2

35

31

30

(32)

2.6

27

20

21

(23)

3.8

7

9

10

(9)

1.5

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

507

430

484

(474)

39.5

466

276

298

(347)

103.9

317

330

301

(316)

14.5

152

157

132

(147)

13.2

2206

2210

2107

(2174)

58.3

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

 

 

Table 4. Test Results: Experiment 2 – With Metabolic Activation

 

Test Period

From: 21 June 2010

To: 24 June 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

107

118

(109)

8.2#

16

18

20

(18)

2.0

40

34

43

(39)

4.6

42

27

24

(31)

9.6

20

11

9

(13)

5.9

+

50

99

108

85

(97)

11.6

11

11

13

(12)

1.2

37

37

37

(37)

0.0

26

24

32

(27)

4.2

15

19

10

(15)

4.5

+

150

81

82

95

(86)

7.8

11

9

22

(14)

7.0

44

42

35

(40)

4.7

37

34

22

(31)

7.9

23

13

9

(15)

7.2

+

500

99

108

136

(114)

19.3

19

13

16

(16)

3.0

33

36

35

(35)

1.5

30

23

26

(26)

3.5

13

7

7

(9)

3.5

+

1500

101

76

75

(84)

14.7

14

9

15

(13)

3.2

32

41

34

(36)

4.7

25

25

30

(27)

2.9

10

11

10

(10)

0.6

+

5000

93

77

84

(85)

8.0

14

9

13

(12)

2.6

40

41

41

(41)

0.6

23

31

29

(28)

4.2

9

12

10

(10)

1.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

592

675

673

(647)

47.4

145

132

152

(143)

10.1

299

262

321

(294)

29.8

188

198

246

(211)

31.0

157

166

161

(161)

4.5

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

The test material was considered to be non-mutagenic with and without metabolic activation and under the conditions of this test.
Executive summary:

Introduction.

The method conforms 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, TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five 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 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) 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 no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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.