Bromobenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA with and without metabolic activation

Link to relevant study records

Reference

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:

22 Nov - 05 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his and trp operon

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II:
Strain TA 100: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
All remaining strains: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Solvent used: DMSO
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 30 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains, except of strain TA 1537

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: none
Water solubility: not soluble
Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate in the presence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment I in the presence of S9 mix.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment I: TA 100 with and without S9 mix: 2500 to 5000 µg/plate
WP2 uvrA with S9 mix: 5000 µg/plate
Experiment II:
without S9 mix: TA 1535, TA 98, TA 100; WP2 uvrA w: 5000 µg/plate
with S9 mix: TA 1535: 1000 - 5000 µg/plate
TA 98: 5000 µg/plate
TA 100: 333 - 5000 µg/plate

Remarks on result:

other:

Remarks:

the test item did not induce gene mutations.

Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMSO		12 ± 4	11 ± 1	28 ± 6	182 ± 0	29 ± 2
Activation	Untreated		11 ± 3	16 ± 2	27 ± 7	201 ± 7	43 ± 2
	Art. 801786	3 µg	12 ± 4	12 ± 5	35 ± 5	205 ± 18	27 ± 6
	(Brombenzene)	10 µg	13 ± 1	12 ± 3	32 ± 5	186 ± 13	39 ± 5
		33 µg	10 ± 4	12 ± 6	23 ± 3	191 ± 16	31 ± 5
		100 µg	14 ± 3	13 ± 6	30 ± 11	205 ± 12	40 ± 5
		333 µg	10 ± 4	12 ± 4	37 ± 8	153 ± 17	36 ± 4
		1000 µg	13 ± 3	10 ± 1	25 ± 4	120 ± 14	41 ± 9
		2500 µg	13 ± 1	8 ± 2	16 ± 0	82 ± 3	25 ± 5
		5000 µg	12 ± 2	7 ± 4	16 ± 6	67 ± 18	20 ± 5
	NaN3	10 µg	1437 ± 130			2691 ± 32
	4-NOPD	10 µg			357 ± 61
	4-NOPD	50 µg		77 ± 9
	MMS	2.0 µL					1115 ± 55
With	DMSO		14 ± 7	10 ± 1	36 ± 6	155 ± 30	46 ± 0
Activation	Untreated		13 ± 6	14 ± 6	43 ± 6	171 ± 56	51 ± 3
	Art. 801786	3 µg	11 ± 4	12 ± 4	28 ± 3	156 ± 32	37 ± 7
	(Brombenzene)	10 µg	12 ± 3	14 ± 2	34 ± 3	141 ± 21	40 ± 6
		33 µg	13 ± 3	13 ± 3	27 ± 9	177 ± 14	50 ± 8
		100 µg	10 ± 6	10 ± 2	39 ± 7	171 ± 21	46 ± 7
		333 µg	10 ± 2	13 ± 1	38 ± 7	145 ± 9	49 ± 4
		1000 µg	10 ± 1	11 ± 3	41 ± 4	124 ± 16	47 ± 5
		2500 µg	11 ± 2^P M	10 ± 2^P M	32 ± 1^P	46 ± 9^P	40 ± 7^P
		5000 µg	11 ± 4^P M	14 ± 2^P M	34 ± 7^P M	24 ± 5^P M	15 ± 2^P M
	2-AA	2.5 µg	505 ± 18	154 ± 12	3562 ± 677	4545 ± 663
	2-AA	10.0 µg					403 ± 40

Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1535 Revertant Colony Counts (Mean ±SD)	TA 1537 Revertant Colony Counts (Mean ±SD)	TA 98 Revertant Colony Counts (Mean ±SD)	TA 100 Revertant Colony Counts (Mean ±SD)	WP2 uvrA Revertant Colony Counts (Mean ±SD)
Without	DMSO		14 ± 4	10 ± 5	26 ± 3	131 ± 5	43 ± 10
Activation	Untreated		9 ± 2	18 ± 2	23 ± 4	194 ± 1	43 ± 8
	Art. 801786	10 µg				119 ± 16
	(Brombenzene)	33 µg	13 ± 2	11 ± 3	24 ± 4	122 ± 16	38 ± 5
		100 µg	14 ± 2	14 ± 3	32 ± 4	114 ± 6	36 ± 1
		333 µg	11 ± 1	11 ± 2	25 ± 2	84 ± 1	43 ± 9
		1000 µg	11 ± 5	12 ± 4	18 ± 4	62 ± 12	33 ± 9
		2500 µg	10 ± 6	8 ± 2	19 ± 3	73 ± 11	29 ± 6
		5000 µg	1 ± 1	7 ± 1	6 ± 1	51 ± 7	10 ± 1
	NaN3	10 µg	1610 ± 69			2364 ± 240
	4-NOPD	10 µg			450 ± 28
	4-NOPD	50 µg		89 ± 8
	MMS	2.0 µL					696 ± 48
With	DMSO		10 ± 1	11 ± 5	31 ± 4	123 ± 5	65 ± 4
Activation	Untreated		15 ± 5	15 ± 6	39 ± 4	184 ± 28	56 ± 11
	Art. 801786	10 µg				112 ± 9
	(Brombenzene)	33 µg	13 ± 3	9 ± 3	39 ± 7	117 ± 10	54 ± 13
		100 µg	11 ± 5	9 ± 3	34 ± 7	98 ± 13	51 ± 7
		333 µg	9 ± 2	9 ± 4	26 ± 2	53 ± 6	45 ± 11
		1000 µg	4 ± 1	10 ± 2	33 ± 9	51 ± 7	42 ± 10
		2500 µg	3 ± 2	11 ± 2	18 ± 6	45 ± 6	30 ± 8
		5000 µg	1 ± 1	11 ± 1	11 ± 3	14 ± 5	30 ± 8
	2-AA	2.5 µg	392 ± 7	92 ± 19	5138 ± 73	4310 ± 254
	2-AA	10.0 µg					363 ± 54

Key to Plate Postfix Codes:              

P: Precipitate

M: Manuel Count

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, Art. 801786 (Brombenzene) is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The test item Art. 801786 (Brombenzene) was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                

TA 100:                                           10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
all remaining strains:                       33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate in the presence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 2500 to 5000 µg/plate in experiment I in the presence of S9 mix.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

 

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	5000	1000 – 5000
TA 1537	/	/	/	/
TA 98	/	/	5000	5000
TA 100	2500 – 5000	2500 – 5000	5000	333 – 5000
WP2 uvrA	/	5000	5000	/

/ = no toxic effect (induction factor>0.5)

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Art. 801786 (Brombenzene) at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria

Mutagenicity of bromobenzene was tested in a GLP-compliant bacterial reverse mutation assay according to OECD TG 471 performed with a standard battery of Salmonella typhimurium tester strains including TA 1535, TA 1537, TA 98 and TA 100 and WP2uvrA bacterial cells (reference 7.6.1-1). Based on the results of a preliminary cytotoxicity test, the highest concentration of 5000 µg/plate was tested in all strains. Bromobenzene did not exhibit mutagenic properties in the absence or presence of metabolic activation. Toxicity indicated by reduced number of revertants was observed in all strains at least at the highest applied dose except for TA 1537 in at least one experiment. Positive control substances induced a distinct increase in the number of revertants in all strains with and without metabolic activation thereby proving validity of the assay. Based on the results of the conducted study, bromobenzene is not considered to exhibit mutagenic properties in bacterial cells.

Justification for classification or non-classification

Based on the available data on genetic toxicity, there is no indication that bromobenzene induces genetic toxicity in bacteria. However, no final decision on classification for genetic toxicity according to Regulation (EC) 1272/2008 can be made, as no information on chromosomal aberration and mutagenicity in mammalian cells/in vivo is available.