4-bromobenzyl alcohol

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:

20 October 2017 - 20 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-bromobenzyl alcohol

EC Number:

212-851-7

EC Name:

4-bromobenzyl alcohol

Cas Number:

873-75-6

Molecular formula:

C7H7BrO

IUPAC Name:

(4-bromophenyl)methanol

Method

Target gene:

his/trp

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

uvrA

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:
Strains TA 1535 and TA 98 (+/- S9 mix): 33; 100; 333; 1000; and 2500; and 5000 µg/plate
Strains TA 1537, TA100 and WP2 uvrA (+/- S9 mix): 10; 33; 100; 333; 1000; and 2500 µg/plate

Experiment IIa:
Strains TA 1537 (+S9 mix) and WP2 uvrA (-S9 mix): 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.

Controls

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: 60 min
- Exposure duration: 72 h

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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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:

cytotoxicity

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 under all experimental conditions. However, no precipitation of the test item occurred up to the highest investigated dose in the overlay agar on the incubated agar plates in this study.
Other confounding effects: none

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviation

ADDITIONAL INFORMATION ON CYTOTOXICITY: Bacteriotoxicity indicated by reduced background growth on the plates was observed from about 2500 µg/plate onward depending on the experimental condition. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in almost all strains from about 2500 µg/plate onward.

Any other information on results incl. tables

Table 1: 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		11 ± 4	8 ± 1	26 ± 4	155 ± 7	41 ± 5
Activation	Untreated		10 ± 4	8 ± 3	31 ± 3	163 ± 7	41 ± 9
	Test item	3 µg	9 ± 3	9 ± 2	20 ± 3	133 ± 13	45 ± 14
		10 µg	7 ± 3	8 ± 3	20 ± 3	133 ± 6	40 ± 8
		33 µg	9 ± 5	11 ± 1	23 ± 4	137 ± 3	47 ± 10
		100 µg	10 ± 1	9 ± 3	31 ± 10	141 ± 14	45 ± 11
		333 µg	8 ± 1	10 ± 1	19 ± 6	142 ± 12	40 ± 7
		1000 µg	12 ± 1	11 ± 2	20 ± 2	137 ± 15	29 ± 2
		2500 µg	10 ± 3	5 ± 1^R	15 ± 4^R	74 ± 8	22 ± 7
		5000 µg	7 ± 2^R	1 ± 1^M R	13 ± 2^M R	21 ± 2^R	11 ± 2
	NaN3	10 µg	1290 ± 30			2471 ± 93
	4-NOPD	10 µg			318 ± 26
	4-NOPD	50 µg		125 ± 12
	MMS	2.0 µL					1025 ± 28
With	DMSO		12 ± 4	11 ± 4	32 ± 7	94 ± 19	44 ± 8
Activation	Untreated		12 ± 3	9 ± 3	34 ± 7	127 ± 14	54 ± 5
	Art. 841647	3 µg	12 ± 5	11 ± 4	33 ± 3	97 ± 10	52 ± 5
	(4-bromobenzyl	10 µg	11 ± 5	10 ± 4	30 ± 7	124 ± 11	52 ± 9
	alcohol)	33 µg	14 ± 5	10 ± 3	33 ± 8	120 ± 4	58 ± 13
		100 µg	9 ± 2	10 ± 1	37 ± 5	125 ± 8	51 ± 1
		333 µg	13 ± 4	15 ± 5	32 ± 10	121 ± 20	43 ± 4
		1000 µg	11 ± 5	13 ± 3	35 ± 6	128 ± 22	36 ± 1
		2500 µg	10 ± 3	9 ± 2^R	28 ± 3	44 ± 12^R	25 ± 5
		5000 µg	3 ± 2	4 ± 1^R M	9 ± 2^M R	9 ± 2^M R	11 ± 3
	2-AA	2.5 µg	464 ± 8	187 ± 13	4795 ± 440	3459 ± 236
	2-AA	10.0 µg					524 ± 5

Table 2: 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		11 ± 1	11 ± 5	29 ± 11	116 ± 23	27 ± 9
Activation	Untreated		12 ± 5	9 ± 4	24 ± 8	212 ± 11	37 ± 4
	Art. 841647	10 µg		10 ± 5		134 ± 15	28 ± 2
	(4-bromobenzyl	33 µg	11 ± 3	8 ± 3	24 ± 7	137 ± 4	28 ± 2
	alcohol)	100 µg	8 ± 2	9 ± 4	24 ± 4	137 ± 5	25 ± 8
		333 µg	12 ± 2	9 ± 4	26 ± 6	121 ± 6	22 ± 6
		1000 µg	11 ± 1	6 ± 1	23 ± 1	104 ± 9	19 ± 6
		2500 µg	11 ± 5	4 ± 2^R	18 ± 3	26 ± 9^R	15 ± 4
		5000 µg	7 ± 2^R		6 ± 3^M R
	NaN3	10 µg	1051 ± 33			1676 ± 82
	4-NOPD	10 µg			366 ± 22
	4-NOPD	50 µg		177 ± 4
	MMS	2.0 µL					812 ± 120
With	DMSO		10 ± 3	11 ± 1	36 ± 6	104 ± 1	37 ± 4
Activation	Untreated		14 ± 2	13 ± 2	41 ± 2	180 ± 23	43 ± 5
	Test item	10 µg		9 ± 4		95 ± 12	40 ± 13
		33 µg	7 ± 3	12 ± 5	37 ± 5	108 ± 6	37 ± 5
		100 µg	7 ± 4	10 ± 4	34 ± 10	116 ± 9	44 ± 11
		333 µg	6 ± 3	11 ± 1	27 ± 1	106 ± 23	33 ± 11
		1000 µg	7 ± 2	10 ± 1	40 ± 7	58 ± 11^R	38 ± 11
		2500 µg	4 ± 1^M R	8 ± 3^R	6 ± 2	4 ± 1^M R	16 ± 4^R
		5000 µg	0 ± 1^M R		1 ± 1^M R
	2-AA	2.5 µg	374 ± 38	116 ± 7	2296 ± 674	1785 ± 123
	2-AA	10.0 µg					421 ± 26

Table 3: Summary of Experiment IIa

Metabolic Activation	Test Group	Dose Level (per plate)	TA 1537 Revertant Colony Counts (Mean ± SD)	WP2 uvrA Revertant Colony Counts (Mean ± SD)
Without	DMSO			27 ± 4
Activation	Untreated			36 ± 6
	Test item	33 µg		36 ± 4
		100 µg		31 ± 11
		333 µg		34 ± 2
		1000 µg		27 ± 6
		2500 µg		17 ± 4
		5000 µg		7 ± 1^R
	MMS	2.0 µL		911 ± 32
With	DMSO		13 ± 3
Activation	Untreated		12 ± 3
	Test item	33 µg	11 ± 2
		100 µg	11 ± 2
		333 µg	13 ± 2
		1000 µg	12 ± 2
		2500 µg	4 ± 2
		5000 µg	1 ± 1
	2-AA	2.5 µg	123 ± 14

Key to Plate Postfix Codes:              

M: Manuel Count

R:  Reduced Background growth

Key to positive controls:

NaN3: sodium azide

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

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Table 4: Bacteriotoxicity

Strain	Exp.I without S9 mix	Exp. I with S9 mix	Exp. II without S9 mix	Exp. II with S9 mix	Exp. IIa without S9 mix	Exp. IIa with S9 mix
TA 1535	/	5000	/	2500 - 5000	-	-
TA 1537	5000	5000	2500	/	-	2500 - 5000
TA 98	/	5000	5000	2500 - 5000	-	-
TA 100	5000	5000	2500	2500	-	-
WP2 uvrA	5000	5000	/	2500	5000	-

/ = no toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5)

- = not performed

Applicant's summary and conclusion

Conclusions:

In an in vitro bacterial reverse mutation assay according to OECD 471 the test item did not induce gene mutations.

Executive summary:

The potential of the test item to induce gene mutations was assessed according to OECD Guideline 471 using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in three independent experiments with and without liver microsomal activation (phenobarbital/beta-naphthoflavone induced rat liver S9 mix). Each concentration and the controls were tested in triplicate. The test item was tested at the concentration 3 - 5000 µg/plate (pre-Experiment/Experiment I), 33 - 5000 µg/plate (Experiment II, Strains TA 1535, TA 98), 10 - 2500 µg/plate (Experiment II, Strains TA 1537, TA 100, E.coli WP2uvrA) and 33 - 5000 µg/plate (Experiment IIa). The test item precipitated in the overlay agar in the test tubes at 5000 µg/plate under all experimental conditions. However, no precipitation of the test item occurred up to the highest investigated dose in the overlay agar on the incubated agar plates in this study. The plates incubated with the test item showed reduced background growth as indication of bacteria toxicity from about 2500 µg/plate onward depending on the experimental condition. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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. Positive, vehicle and negative controls were valid. In conclusion, 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, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.