Bis(2-ethylhexyl) tetrabromophthalate

Genetic toxicity: in vitro

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

Test material information

Constituent 1

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

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

Reference substance name:

bis(2-ethylhexyl) 3,4,5-tribromophthalate

Molecular formula:

C24H35Br344

IUPAC Name:

bis(2-ethylhexyl) 3,4,5-tribromophthalate

impurity 3

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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 results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.