Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test according to OECD guideline 471, 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.

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

In a further Ames test according to OECD guideline 471, the compound RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) was examined for mutagenic activity in five histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 1538, TA 100, TA 1535 and TA 1537, using pour-plate assays. Each test, in each strain, was conducted on two separate occasions. The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) from 50 to 5000 ug per plate. RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) was negative (devoid of mutagenic activity) under the conditions of the test.

In an in vitro Mammalian Chromosome Aberration Test according to OECD guideline 473, RC9927 (bis(2-ethylhexyl)tetrabromophthalate) was positive and showed evidence of weak clastogenic activity at 1000 ug/ml.

The available HPRT assay according to OECD guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) with bis(2-ethylhexyl)tetrabromophthalate was negative. No substantial and reproducible dose dependent increase of the mutation frequency was observed.

Link to relevant study records

Referenceopen allclose all

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
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
Specific details on test material used for the study:
Storage Conditions: Room temperature, in the dark
Expiry Date: 12 May 2018
Target gene:
Histidine locus in S. typhimurium and tryptophan locus in E.coli.
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.
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.
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).

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

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.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The compound RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) was examined for mutagenic activity in five histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 1538, TA 100, TA 1535 and TA 1537, using pour-plate assays. Each test, in each strain, was conducted on two separate occasions. The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) from 50 to 5000 ug per plate.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100, TA 1535 , TA 1537 and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Test 1 and test 2: 0, 50, 158, 500, 1580, 5000 µg per plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1535 , TA 1537 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: S. typhimurium TA 98, TA 100, TA 1535 , TA 1537 and TA 1538

No increases in reversion to prototrophy were obtained with any of the five bacterial strains following exposure to RC9927 (bis(2-ethylhexyl)tetrabromophthalate) at levels from 50 to 5000 µg per plate, either in the presence or absence of S-9 mix.

Marked increases in the number of revertant colanies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

Conclusions:
Interpretation of results: negative
Executive summary:

The compound RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) was examined for mutagenic activity in five histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 1538, TA 100, TA 1535 and TA 1537, using pour-plate assays. Each test, in each strain, was conducted on two separate occasions. The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a range of levels of RC 9927 from 50 to 5000 ug per plate.

No increases in reversion to prototrophy were obtained with any of the five bacterial strains following exposure to RC9927 (bis(2-ethylhexyl)tetrabromophthalate) at levels from 50 to 5000 µg per plate, either in the presence or absence of S-9 mix.

Marked increases in the number of revertant colanies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine and sodium azide when examined under similar conditions.

RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) was devoid of mutagenic activity under the conditions of the test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
The effects on chromosomal structure of a 24-hour exposure to RC9927 (bis(2-ethylhexyl)tetrabromophthalate) were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activating system (S-9 mix) during the first two hours of exposure. Treatments were established by the addition of test solutions (in dimethyl sulphoxide : DMSO) to 48 hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 ug/ml), three hours before the cells were harvested; slides were then prepared for microscopic analysis.
Mitotic indices were calculated for each culture: these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated; these aberrant cell frequencies were calculated both including and excluding gap-type aberrations.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
no data
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Human peripheral blood was obtained by venupuncture froma healthy, male, human volunteer
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix
Test concentrations with justification for top dose:
Main test: 0, 40, 200, or 1000 µg/ml test substance
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
other: chlorambucil
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

A preliminary test was first performed to investigate the toxicity of RC9927 (bis(2-ethylhexyl)tetrabromophthalate) to dividing lymphocytes. In this test, RC9927 (bis(2-ethylhexyl) tetrabromo- phthalate) was tested up to the maximum practicable concentration of 1000 ug/ml.

No evidence of toxicity was seen : consequently the concentrations selected for use in the subsequent cytogenetic test were 40, 200 and 1000 ug/ml.

This main test also incorporated vehicle and positive (cyclophosphamide and chlorambucil) control cultures.

No evidence of toxicity to dividing lymphocytes was seen in any RC9927-treated culture, with or without the inclusion of S-9 mix.

In solvent control cultures, the frequencies of aberrant cells ranged from 1 to 3%. Cultures treated with RC9927 at 40 and 200 ug/ml had aberrant cell frequencies within this range (except for a single, non-activated culture treated at 40 ug/ml, which had a frequency of 4%), but those treated at 1000 ug/ml had frequencies of 3-5%.

For each treatment (excepting chlorambucil, which was not tested with S-9 mix), the effect of coincubation with S-9 mix on aberrant cell frequency was examined by statistical analysis. Where there was no significant difference between activated and non-activated cultures which were otherwise similarly treated, the data were pooled in further statistical analysis. For this reason, results from activated and non-activated cultures were pooled within each group. Statistical analysis was performed on the frequencies of aberrant metaphases, both including and excluding gap type aberrations.

No statistically significant increases in the frequency of aberrant cells over controls were recorded for cultures treated with RC9927 (bis(2-ethylhexyl)tetrabromophthalate) at 40 and 200 µg/ml (p > 0.05); this was true whether gap-type aberrations were included in or excluded from analysis. However, at 1000 µg/ml statistically significant increases in aberrant cell frequencies were seen both including and excluding gaps (0.001 < p < 0.01 in both cases).

The known clastogens, cyclophosphamide and chlorambucil, induced significant increases in chromosomal darnage over the vehicle controls (p < 0.001 in both cases; as expected, cyclophosphamide was active only in the presence of S-9 mix).

Conclusions:
Interpretation of results: positive
Executive summary:

The effects on chromosomal structure of a 24-hour exposure to RC9927 (bis(2-ethylhexyl)tetrabromophthalate) were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activating system (S-9 mix) during the first two hours of exposure. Treatments were established by the addition of test solutions (in dimethyl sulphoxide : DMSO) to 48 hour cultures established from whole, human blood. Cell divisionwas arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 ug/ml), three hours before the cells were harvested; slides were then prepared for microscopic analysis.

Mitotic indices were calculated for each culture: these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated; these aberrant cell frequencies were calculated both including and excluding gap-type aberrations.

No statistically significant increases in the frequency of aberrant cells over controls were recorded for cultures treated with RC9927 (bis(2-ethylhexyl)tetrabromophthalate) at 40 and 200 µg/ml (p > 0.05); this was true whether gap-type aberrations were included in or excluded from analysis. However, at 1000 µg/ml statistically significant increases in aberrant cell frequencies were seen both including and excluding gaps (0.001 < p < 0.01 in both cases).

Under the conditions of the test, RC9927 (bis(2-ethylhexyl)tetrabromophthalate) showed evidence of weak clastogenic activity at 1000 ug/ml. The sensitivity of the test procedure, and the metabolic activity of the S-9 mix employed, were demonstrated by the clear responses to the positive control agents.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The study was performed to investigate the potential of Bis(2-ethylhexyl)tetrabromophthalate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
Identity: Bis(2-ethylhexyl)tetrabromophthalate
Batch No.: 12128E71
Purity: 90.6%; dose calculation adjusted to purity
Molecular Weight: 706.1 g/mol
Storage: At room temperature
Target gene:
HPRT locus in V79 cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Additional strain / cell type characteristics:
other: he cells have a stable karyothype with a modal chromosome number of 22
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
exposure S9 concentrations in µg/mL
period mix
Experiment I
4 hours - 3.0 9.0 18.0 54.0ps 162.0ps 486.0ps
4 hours + 3.0 9.0 18.0 54.0ps 162.0ps 486.0ps
Experiment II
24 hours - 3.0 9.0 18.0 54.0ps 162.0 ps 486.0 ps
4 hours + 3.0 9.0 18.0 54.0ps 162.0 ps 486.0 ps
ps = phase separation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The maximum test item concentration of 5800 µg/mL used in the pre-experiment with and without metabolic activation was equal to approximately 5000 µg/mL of the pure substance. The test item was dissolved in acetone. The dose range of the main experiments was limited by phase separation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusions:
Interpretation of results: negative
Executive summary:

The study was performed to investigate the potential of bis(2-ethylhexyl)tetrabromophthalate to induce gene muutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The maximum test item concentration of 5800 µg/mL used in the pre-experiment with and without metabolic activation was equal to approximately 5000 µg/mL of the pure substance. The test item was dissolved in acetone. The dose range of the main experiments was limited by phase separation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, bis(2-ethylhexyl)tetrabromophthalate is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In a study according to OECD guideline 474 (Mammalian Erythrocyte Micronucleus Test), the effect of RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) on chromosome structure in bone marrow cells was investigated following acute intraperitoneal and sub-acute dermal administration to mice. Chromosome damage was measured indirectly by counting micronuclei.

Under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice, after either acute intraperitoneal or sub-acute dermal administration of RC 9927 (bis(2-ethylhexyl)tetrabromophthalate). Therefore, the test is negative.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
The effect of RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) on chromosome structure in bone marrow cells was investigated following acute intraperitoneal and sub-acute dermal administration to mice. Chromosome damage was measured indirectly by counting micronuclei.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Route of administration:
dermal
Duration of treatment / exposure:
Intraperitoneal route: animals were killed 48 or 72 hours after treatment.
Dermal route: animals were treated 5 days and killed 18 or 48 hours after the final treatment.
Frequency of treatment:
Intraperitoneal: single treatment.
Dermal: 5 separate treatments (24 hour intervals).
Post exposure period:
None
Remarks:
Doses / Concentrations:
0, 250, 500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
main micronucleus test - intraperitoneal route
Remarks:
Doses / Concentrations:
0, 2000 mg/kg bw
Basis:
nominal conc.
main micronucleus test - dermal application
No. of animals per sex per dose:
Main Micronucleus Test - intraperitoneal route
Group Treatment Dosage Number Animal
(mg/kg) of mice numbers
1 Corn oil - 15M 17 - 31
15 F 62 - 76
2 RC 9927 80 5M 32 - 36
5F 7 - 81
3 RC 9927 400 5M 37 - 41
5F 82 - 86
4 RC 9927 2 000 15M 42 - 56
15F 87 - 101
5 Chlorambucil 30 5M 57 - 61
5F 102 - 106

Main Micronucleus Test - dermal application
Group Treatment Dosage Number Animal
(mg/kg) of mice numbers
6 Corn oil - 10M 107 - 116
10F 127 136
7 RC 9927 2000 10M 117 - 126
10F 137 - 146

Control animals:
yes
Tissues and cell types examined:
erythrocytes of bone marrow cells
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Frequencies of micronucleated polychromatic erythrocytes in animals exposed to RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) for 24, 48 or 72 hours via the intraperitoneal route were similar to those in concurrent controls. This lack of treatment-related effect was confirmed by statistical analysis.

Frequencies of micronucleated polychromatic erythrocytes in animals treated dermally for 5 days with RC 9927 (bis(2-ethylhexyl)- tetrabromophthalate) and sacrificed 18 or 48 hours after the final treatment were also similar to those in concurrent controls. Again, statistical analysis confirmed that there was no significant increase in the frequencies of micronucleated polychromatic cells in RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) treated groups compared to concurrent vehicle control groups at either termination time.

Statistically significant increases over controls were seen in positive control group animals given chlorambucil at 30 mg/kg (p < 0.01).

Conclusions:
Interpretation of results: negative
Executive summary:

The effect of RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) on chromosome structure in bone marrow cells was investigated following acute intraperitoneal and sub-acute dermal administration to mice. Chromosome damage was measured indirectly by counting micronuclei.

Frequencies of micronucleated polychromatic erythrocytes in animals exposed to RC 9927 (bis(2-ethylhexyl)tetrabromophthalate) for 24, 48 or 72 hours via the intraperitoneal route were similar to those in concurrent controls. This lack of treatment-related effect was confirmed by statistical analysis.

Frequencies of micronucleated polychromatic erythrocytes in animals treated dermally for 5 days with RC 9927 (bis(2-ethylhexyl)- tetrabromophthalate) and sacrificed 18 or 48 hours after the final treatment were also similar to those in concurrent controls. Again, statistical analysis confirmed that there was no significant increase in the frequencies of micronucleated polychromatic cells in RC 9927 treated groups compared to concurrent vehicle control groups at either termination time.

Statistically significant increases over controls were seen in positive control group animals given chlorambucil at 30 mg/kg (p < 0.01).

Under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice, after either acute intraperitoneal or sub-acute dermal administration of RC 9927 (bis(2-ethylhexyl)tetrabromophthalate).

Therefore, the test is negative.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In 2 Ames tests and the V79/HGPRT test bis(2-ethylhexyl)tetrabromophthalate) was negative. In the in-vitro CA (chromosome aberration test) at the highest applied dose a statistically significant increases in aberrant cell frequencies were seen.

However, in the in-vivo MNT, bis(2-ethylhexyl)tetrabromophthalate was negative. Therefore, as an overall result, bis(2-ethylhexyl)tetrabromophthalate is judged as negative, as the in-vivo MNT test has a higher significance and validity than the in-vitro chromosome aberration assay.

Endpoint Conclusion: No adverse effect observed (negative).

Justification for classification or non-classification

Based on a weight-of-evidence consideration, due to the negative results in the 2 Ames tests, the V79/HGPRT test and the in-vivo MNT (which has a higher significance and validity as the positive CA test), genotoxicity is judged as negative.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.