Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Dec 2009 - 01 Feb 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom, UK

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene]

EC Number:

306-832-3

EC Name:

1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene]

Cas Number:

97416-84-7

Molecular formula:

C23H24Br8O2

IUPAC Name:

1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene]

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

his operon (S. typhimurium strains)
trp operon (E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with 80 mg/kg bw/day phenobarbitone and 100 mg/kg bw/day beta-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate, with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was soluble in DMSO at 50 mg/mL

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

See 'Details on test system and conditions' for concentrations and strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, experiment 1) and preincubation (experiment 2)

DURATION
- Preincubation period: 20 min (experiment 2)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replication each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in growth of bacterial background lawn

OTHER:
- positive control substances used: N-ethyl-N'-nitro-N-nitrosoguanidine (2 μg/plate in DMSO, -S9, WP 2urvA-; 3 μg/plate, -S9, TA 100; 5 μg/plate, -S9, TA 1535); 9-aminoacridine (80 μg/plate in DMSO, -S9, TA 1537); 4-nitroquinoline-1-oxide (0.2 μg/plate in DMSO, -S9, TA 98); 2-aminoanthracene (1 μg/plate in DMSO, +S9, TA 100; 2 μg/plate, +S9 TA 1535 and TA 1537; 10 μg/plate, +S9, WP 2urvA-); benzo-a-pyrene (5 μg/plate in DMSO, +S9, TA 98)

Evaluation criteria:

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

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

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

Remarks:

precipitation was observed from 1500 μg/plate in experiment 1 and from 500 μg/plate in experiment 2, with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitate was observed at concentrations at and above 1500 μg/plate in experiment 1 and concentrations at and above 500 μg/plate in experiment 2, with and without metabolic activation, respectively. This did not affect the scoring of revertant colonies.

RANGE-FINDING/SCREENING STUDIES: A toxicity screening study was performed to determine the toxicity of the test substance. 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate test substance was tested on TA 100 and E. coli WP2 uvrA using the plate incorporation method. Precipitation of the test material was observed at 5000 μg/plate. A range-finding study (plate incorporation method) was performed on all strains using dose levels of 50 - 5000 μg/plate. As the results for all the tested dose levels were valid, the results were used as part of the main study (experiment 1).

COMPARISON WITH HISTORICAL CONTROL DATA: yes, the results of the vehicle and positive controls fell within the range of the historical control data (see Table 3 under 'Any other information on results incl. tables')

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed at any dose level.

Any other information on results incl. tables

Table 1. Test results of experiment 1 (plate incorporation method)

With or without S9-mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvR	TA98	TA1537
–	DMSO	134 ± 5.7	27 ± 2.3	28 ± 0.6	26 ± 2.0	11 ± 5.0
–	50	136 ± 4.4	25 ± 0.0	26 ± 2.5	27 ± 0.6	14 ± 1.5
–	150	131 ± 6.4	29 ± 3.8	25 ± 6.7	24 ± 4.5	11 ± 3.8
–	500	115 ± 21.7	28 ± 1.5	27 ± 1.2	25 ± 1.0	17 ± 1.0
–	1500*	106 ± 18.0	25 ± 4.2	22 ± 4.7	22 ± 3.6	10 ± 2.6
–	5000*	116 ± 5.1	32 ± 2.1	16 ± 1.7	19 ± 1.2	10 ± 2.1
Positive controls, -S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	429 ± 43.7	514 ± 43.1	726 ± 77.5	133 ± 11.0	884 ± 92.2
+	DMSO	102 ± 11.6	12 ± 2.5	25 ± 3.5	28 ± 2.3	12 ± 3.2
+	50	109 ± 10.4	12 ± 1.5	27 ± 0.6	28 ± 3.5	15 ± 1.0
+	150	106 ± 3.2	12 ± 1.0	29 ± 0.6	28 ± 1.2	11 ± 4.0
+	500	97 ± 1.5	14 ± 1.7	26 ± 5.0	25 ± 2.1	13 ± 2.6
+	1500*	101 ± 8.1	14 ± 1.2	24 ± 3.5	26 ± 2.6	11 ± 2.0
+	5000*	93 ± 3.0	13 ± 2.6	26 ± 2.5	27 ± 1.5	9 ± 2.6
Positive controls, +S9	Name	2AA	2AA	2AA	BaP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1547 ± 34.6	223 ± 24.5	367 ± 41.1	218 ± 74.1	299 ± 29.5

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

4NQO: 4-nitroquinoline N-oxide

9AA:9-aminocridine

2AA: 2-amino-anthracene

BaP: benzo-a-pyrene

*: precipitation

 

Table 2. Test results of experiment 2 (plate incorporation method)

With or without S9-mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2 uvR	TA98	TA1537
–	DMSO	102 ± 12.7	24 ± 2.5	22 ± 6.1	18 ± 4.4	16 ± 2.5
–	50	85 ± 12.4	24 ± 5.0	21 ± 2.6	15 ± 2.3	13 ± 1.2
–	150	94 ± 7.1	24 ± 2.9	18 ± 2.5	17 ± 5.1	13 ± 1.7
–	500*	90 ± 10.0	23 ± 4.0	21 ± 0.6	20 ± 2.1	15 ± 6.1
–	1500*	94 ± 5.1	26 ± 3.5	22 ± 1.5	20 ± 2.6	14 ± 1.5
–	5000*	96 ± 4.5	24 ± 2.3	23 ± 3.5	20 ± 2.0	14 ± 2.0
Positive controls, -S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	613 ± 32.2	578 ± 58.0	861 ± 68.6	144 ± 9.5	354 ± 120.8
+	DMSO	99 ± 8.5	16 ± 2.1	28 ± 5.1	27 ± 6.7	14 ± 3.2
+	50	90 ± 4.2	15 ± 1.0	29 ± 1.5	25 ± 4.0	10 ± 1.5
+	150	94 ± 6.7	14 ± 1.5	25 ± 3.6	25 ± 8.2	14 ± 1.5
+	500*	98 ± 12.7	15 ± 1.2	24 ± 3.1	28 ± 1.7	14 ± 2.0
+	1500*	98 ± 2.1	16 ± 3.0	25 ± 3.6	26 ± 4.4	16 ± 2.5
+	5000*	97 ± 4.7	12 ± 1.5	26 ± 1.5	25 ± 2.3	12 ± 1.5
Positive controls, +S9	Name	2AA	2AA	2AA	BaP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	586 ± 96.2	235 ± 12.0	259 ± 36.7	387 ± 51.7	165 ± 8.4

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

4NQO: 4-nitroquinoline N-oxide

9AA:9-aminocridine

2AA: 2-amino-anthracene

BaP: benzo-a-pyrene

*: precipitation

Table 3. Historical controls

Strain	Control	without S9-mix	with S9-mix	Year
		Mean ± SD	Mean ± SD
TA100	Solvent/untreated	94 ± 16.0	87 ± 14.8	2007
TA100	Positive	558 ± 313.5	1231 ± 651.1	2007
TA1535	Solvent/untreated	21 ± 5.2	13 ± 3.6	2007
TA1535	Positive	343 ± 864.2	231 ± 476.5	2007
WP2 uvR	Solvent/untreated	26 ± 5.8	28 ± 6.4	2007
WP2 uvR	Positive	652 ± 707.9	505 ± 535.2	2007
TA98	Solvent/untreated	21 ± 5.5	26 ± 6.0	2007
TA98	Positive	256 ± 289.7	308 ± 280.5	2007
TA1537	Solvent/untreated	11 ± 3.8	13 ± 4.1	2007
TA1537	Positive	1697 ± 1193.6	339 ± 254.4	2007
TA100	Solvent/untreated	105 ± 18.7	101 ± 18.5	2008
TA100	Positive	487 ± 184.7	1138 ± 517.2	2008
TA1535	Solvent/untreated	23 ± 5.3	15 ± 4.8	2008
TA1535	Positive	210 ± 107.7	272 ± 94.9	2008
WP2 uvR	Solvent/untreated	26 ± 5.9	29 ± 7.2	2008
WP2 uvR	Positive	548 ± 231.5	426 ± 273.9	2008
TA98	Solvent/untreated	19 ± 4.6	26 ± 5.3	2008
TA98	Positive	155 ± 70.8	247 ± 178.2	2008
TA1537	Solvent/untreated	13 ± 3.7	13 ± 3.8	2008
TA1537	Positive	930 ± 476.2	279 ± 101.7	2008