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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:
Experimental start date: 18 April 2017 Experimental completion date: 08 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) tetrabromophthalate
EC Number:
247-426-5
EC Name:
Bis(2-ethylhexyl) tetrabromophthalate
Cas Number:
26040-51-7
Molecular formula:
C24H34Br4O4
IUPAC Name:
1,2-bis(2-ethylhexyl) 3,4,5,6-tetrabromobenzene-1,2-dicarboxylate
impurity 1
Chemical structure
Reference substance name:
Benzoic acid, 2,3,4,5-tetrabromo-, 2-ethylhexyl ester
Cas Number:
183658-27-7
Molecular formula:
C15H18Br4O2
IUPAC Name:
Benzoic acid, 2,3,4,5-tetrabromo-, 2-ethylhexyl ester
impurity 2
Chemical structure
Reference substance name:
bis(2-ethylhexyl) 3,4,5-tribromophthalate
Molecular formula:
C24H35Br344
IUPAC Name:
bis(2-ethylhexyl) 3,4,5-tribromophthalate
impurity 3
Chemical structure
Reference substance name:
bis(2-ethylhexyl) 3,4,6-tribromophthalate
Cas Number:
122857-50-5
Molecular formula:
C24H35Br3O4
IUPAC Name:
bis(2-ethylhexyl) 3,4,6-tribromophthalate
Test material form:
liquid
Details on test material:
Batch No.: GS16337E71
Specific details on test material used for the study:
Storage Conditions: Room temperature, in the dark
Expiry Date: 12 May 2018

Method

Target gene:
Histidine locus in S. typhimurium and tryptophan locus in E.coli.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (10% liver S9 in standard co factors)
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were tested

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 µg/plate.
Six concentrations of the test item (15, 50, 150, 500, 1500 and 5000 µg/plate) were tested
Vehicle / solvent:
The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in–house. Acetone was selected as the vehicle. The homogeneity and stability was confirmed for the test item as outlined in the Study Plan in acetone formulations at nominal concentrations of 0.1 and 200 mg/mL.
Furthermore, to support this, homogeneity and stability were previously confirmed in Harlan CCR study 1502500 for concentrations of 0.05 and 500 mg/mL.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100, 5 µg/plate for TA1535
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
80 µg/plate for TA1537
Positive control substance:
9-aminoacridine
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
0.2 µg/plate for TA98
Positive control substance:
other: 4-Nitroquinoline-1-oxide
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
1 µg/plate for TA100, 2 µg/plate for TA1535 and TA1537, 10 µg/plate WP2uvrA
Positive control substance:
other: 2-Aminoanthracene
Remarks:
Presence of S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Remarks:
5 µg/plate for TA98
Positive control substance:
benzo(a)pyrene
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. No correction was made for purity. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots (Maron et al., 1981). All formulations were used within four hours of preparation. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. Analysis was carried out in Experiment 1 to determine the concentration of the maximum test item formulation (50 mg/mL).
During the Dose Formulation Analysis phase, only the maximum dose level in the first experiment (50 mg/mL) was analyzed; the absence of a Dose Formulation Analysis of a mid and low dose level is considered an exception which is thought not to affect the purpose or integrity of the study.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
Eight concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Six test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.05 mL of the test item formulation or solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 ºC for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Due to a hardware failure, Ames study manager and sorcerer system suffered an extended downtime, resulting in manual counts being performed on all of the plates produced for Experiment 2.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item 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 making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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 the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (acetone) 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).
A test item precipitate (globular and light in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

102

 

22

 

28

 

27

 

9

 

88

(89)

27

(26)

33

(32)

19

(24)

3

(8)

78

 

30

 

34

 

25

 

13

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

80

 

23

 

24

 

22

 

19

 

91

(82)

15

(19)

29

(24)

21

(22)

9

(14)

76

 

19

 

20

 

23

 

13

 

 

  Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 20 June 2017

To: 23 June 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

95

113

97

(102)

9.9#

24

20

24

(23)

2.3

36

21

25

(27)

7.8

23

21

22

(22)

1.0

8

18

18

(15)

5.8

1.5 µg

93

87

74

(85)

9.7

27

30

31

(29)

2.1

28

33

27

(29)

3.2

16

25

17

(19)

4.9

14

12

17

(14)

2.5

5 µg

63

64

74

(67)

6.1

29

32

16

(26)

8.5

33

27

28

(29)

3.2

21

24

22

(22)

1.5

11

26

11

(16)

8.7

15 µg

73

61

76

(70)

7.9

17

19

21

(19)

2.0

28

32

27

(29)

2.6

13

28

27

(23)

8.4

19

18

20

(19)

1.0

50 µg

94

71

83

(83)

11.5

30

21

19

(23)

5.9

40

17

17

(25)

13.3

29

15

21

(22)

7.0

12

13

15

(13)

1.5

150 µg

64

72

81

(72)

8.5

21

27

21

(23)

3.5

28

29

31

(29)

1.5

13

29

24

(22)

8.2

11

7

18

(12)

5.6

500 µg

73

72

63

(69)

5.5

30

30

16

(25)

8.1

32

22

22

(25)

5.8

14

22

12

(16)

5.3

12

11

16

(13)

2.6

1500 µg

72

81

81

(78)

5.2

25

27

25

(26)

1.2

31

21

37

(30)

8.1

20

27

23

(23)

3.5

20

10

14

(15)

5.0

5000 µg

80 P

73 P

72 P

(75)

4.4

27 P

27 P

25 P

(26)

1.2

28 P

30 P

37 P

(32)

4.7

21 P

21 P

17 P

(20)

2.3

17 P

10 P

14 P

(14)

3.5

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

509

456

595

(520)

70.1

531

699

676

(635)

91.1

752

744

742

(746)

5.3

241

224

249

(238)

12.8

118

144

178

(147)

30.1

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 20 June 2017

To: 23 June 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

84

92

102

(93)

9.0#

29

21

16

(22)

6.6

43

38

39

(40)

2.6

28

22

25

(25)

3.0

15

17

9

(14)

4.2

1.5 µg

71

66

67

(68)

2.6

17

27

27

(24)

5.8

40

45

33

(39)

6.0

20

22

25

(22)

2.5

13

16

15

(15)

1.5

5 µg

63

66

65

(65)

1.5

26

15

26

(22)

6.4

29

37

34

(33)

4.0

25

18

21

(21)

3.5

13

10

13

(12)

1.7

15 µg

67

69

79

(72)

6.4

29

19

28

(25)

5.5

49

33

21

(34)

14.0

21

30

23

(25)

4.7

15

19

8

(14)

5.6

50 µg

79

73

73

(75)

3.5

26

27

12

(22)

8.4

38

20

32

(30)

9.2

25

24

21

(23)

2.1

23

16

5

(15)

9.1

150 µg

81

76

79

(79)

2.5

27

19

26

(24)

4.4

40

31

29

(33)

5.9

26

23

28

(26)

2.5

6

8

13

(9)

3.6

500 µg

79

93

74

(82)

9.8

26

21

17

(21)

4.5

49

34

35

(39)

8.4

26

27

24

(26)

1.5

14

13

20

(16)

3.8

1500 µg

91

72

79

(81)

9.6

28

27

27

(27)

0.6

44

39

38

(40)

3.2

28

26

25

(26)

1.5

7

13

14

(11)

3.8

5000 µg

71 P

72 P

79 P

(74)

4.4

30 P

29 P

22 P

(27)

4.4

35 P

27 P

32 P

(31)

4.0

31 P

26 P

27 P

(28)

2.6

17 P

13 P

14 P

(15)

2.1

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

575

620

501

(565)

60.1

311

208

232

(250)

53.9

146

167

165

(159)

11.6

208

165

169

(181)

23.8

608

621

618

(616)

6.8

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#            Standard deviation

Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 02 August 2017

To: 05 August 2017

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

83

76

82

(80)

3.8#

13

22

18

(18)

4.5

25

32

32

(30)

4.0

25

17

25

(22)

4.6

18

10

12

(13)

4.2

15 µg

76

87

72

(78)

7.8

16

18

24

(19)

4.2

18

25

37

(27)

9.6

17

19

24

(20)

3.6

18

11

9

(13)

4.7

50 µg

66

77

73

(72)

5.6

19

15

17

(17)

2.0

28

35

26

(30)

4.7

25

21

19

(22)

3.1

12

13

17

(14)

2.6

150 µg

71

70

80

(74)

5.5

19

17

20

(19)

1.5

32

30

26

(29)

3.1

22

21

16

(20)

3.2

13

20

6

(13)

7.0

500 µg

73

71

74

(73)

1.5

19

16

12

(16)

3.5

28

27

27

(27)

0.6

19

20

21

(20)

1.0

12

15

12

(13)

1.7

1500 µg

87

72

81

(80)

7.5

18

12

22

(17)

5.0

31

21

24

(25)

5.1

22

20

17

(20)

2.5

8

9

19

(12)

6.1

5000 µg

74 P

75 P

85 P

(78)

6.1

19 P

22 P

22 P

(21)

1.7

24 P

31 P

19 P

(25)

6.0

23 P

18 P

20 P

(20)

2.5

11 P

13 P

17 P

(14)

3.1

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

594

620

585

(600)

18.2

1113

1166

932

(1070)

122.7

1095

1041

941

(1026)

78.1

240

256

263

(253)

11.8

174

278

249

(234)

53.7

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#         Standard deviation

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 02 August 2017

To: 05 August 2017

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(Acetone)

80

83

80

(81)

1.7#

25

21

12

(19)

6.7

22

29

29

(27)

4.0

28

20

26

(25)

4.2

14

10

16

(13)

3.1

15 µg

80

75

69

(75)

5.5

15

22

13

(17)

4.7

21

24

32

(26)

5.7

30

22

20

(24)

5.3

16

12

16

(15)

2.3

50 µg

80

74

70

(75)

5.0

29

21

11

(20)

9.0

27

28

29

(28)

1.0

31

20

28

(26)

5.7

14

13

9

(12)

2.6

150 µg

69

85

73

(76)

8.3

15

20

20

(18)

2.9

29

24

31

(28)

3.6

18

30

28

(25)

6.4

10

14

15

(13)

2.6

500 µg

78

68

77

(74)

5.5

23

21

18

(21)

2.5

28

26

34

(29)

4.2

25

27

16

(23)

5.9

13

19

18

(17)

3.2

1500 µg

73

88

70

(77)

9.6

14

22

20

(19)

4.2

22

26

31

(26)

4.5

26

25

31

(27)

3.2

13

14

12

(13)

1.0

5000 µg

87 P

77 P

70 P

(78)

8.5

19 P

17 P

23 P

(20)

3.1

28 P

27 P

29 P

(28)

1.0

19 P

29 P

24 P

(24)

5.0

12 P

18 P

16 P

(15)

3.1

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1090

1088

1039

(1072)

28.9

252

266

247

(255)

9.8

210

213

239

(221)

15.9

162

150

144

(152)

9.2

389

367

286

(347)

54.2

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#            Standard deviation

Applicant's summary and conclusion

Conclusions:
Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, 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 OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7) using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co‑factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (Experiment 2, pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 2 was amended, following the results of Experiment 1, and was 15 to 5000 µg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Formulation analysis was carried out in Experiment 1 to determine the concentration of the test item concentration (maximum dose). 

Results

The vehicle (acetone) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). 

A test item precipitate (globular and light in appearance) was noted at 5000 mg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). 

Conclusion

Bis(2-ethylhexyl) tetrabromophthalate (CAS No. 26040-51-7)was considered to be non-mutagenic under the conditions of this test.