Benzofuran-2-yl methyl ketone

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:

3 Feb - 14 Mar 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzofuran-2-yl methyl ketone

EC Number:

216-706-9

EC Name:

Benzofuran-2-yl methyl ketone

Cas Number:

1646-26-0

Molecular formula:

C10H8O2

IUPAC Name:

benzofuran-2-yl methyl ketone

Method

Target gene:

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

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

The pre-experiment is reported as Experiment 1.

Experiment 2:
33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for TA 100 and TA 1535
10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation for remaining strains

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (Experiment 1); pre-incubation (Experiment 2)

DURATION
- Preincubation period: 1 h
- Exposure duration: at least 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered as a mutagenic 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:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 2: -S9: at 5000 µg/plate; +S9: at and above 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1: -S9: at 5000 µg/plate; Exp 2: +/-S9: at and above 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1: +S9: at 5000 µg/plate; Exp 2: +S9: at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1: +S9: at 5000 µg/plate; Exp 2: -S9: at 5000 µg/plate; +S9: at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp 1 and 2: +/- S9: at and above 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes at 5000 μg/plate with and without S9 mix in both experiments. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment 1 at 5000 μg/plate with S9 mix. In Experiment 2 no precipitation of the test susbtance was observed on the incubated agar plates. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment is reported as Experiment I (all strains were tested in the pre-experiment).

ADDITIONAL INFORMATION ON CYTOTOXICITY:The plates incubated with the test substance showed reduced background growth in Experiment 2 in all strains used, except of strain WP2 uvrA. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all strains used.

Any other information on results incl. tables

Table 1: Results of Experiment 1 (plate incorporation).

	Number of revertant colonies (mean of 3 plates ± SD)
Without S9
Test substance (µg/plate)	TA 1535	TA 1537			TA 98	TA 100		WP2 uvr A
DMSO	13 ± 2	8 ± 2			27 ± 2	194 ± 10		33 ± 6
Untreated	12 ± 2	7 ± 3			29 ± 6	208 ± 16		38 ± 5
3	14 ± 2	9 ± 4			26 ± 4	184 ± 22		30 ± 6
10	10 ± 2	8 ± 1			27 ± 5	200 ± 13		33 ± 3
33	9 ± 3	6 ± 1			23 ± 4	181 ± 19		28 ± 4
100	11 ± 4	8 ± 3			28 ± 4	181 ± 18		33 ± 7
333	12 ± 4	10 ± 1			28 ± 7	193 ± 14		30 ± 3
1000	13 ± 3	4 ± 1			26 ± 4	179 ± 8		15 ± 5
2500	12 ± 0	4 ± 2			25 ± 5	166 ± 21		13 ± 3
5000	11 ± 4	3 ± 1			23 ± 4	169 ± 21		14 ± 2
Positive Control	NaN3	4-NOPD			4-NOPD	NaN3		MMS
Dose (µg/plate)	10	50			10	10		2
Number of revertant colonies/plate	930 ± 93	100 ± 18			478 ± 53	2300 ± 55		923 ± 20
With S9
Test substance (µg/plate)	TA 1535		TA 1537		TA 98	TA 100	WP2 uvr A
DMSO	13 ± 3		9 ± 4		37 ± 4	177 ± 8	43 ± 6
Untreated	19 ± 3		9 ± 3		42 ± 8	186 ± 17	46 ± 7
3	9 ± 2		8 ± 4		38 ± 3	183 ± 13	44 ± 11
10	11 ± 2		8 ± 3		40 ± 2	179 ± 6	42 ± 7
33	10 ± 3		7 ± 2		31 ± 2	180 ± 22	44 ± 11
100	12 ± 4		11 ± 3		31 ± 4	188 ± 21	48 ± 7
333	15 ± 1		11 ± 6		41 ± 5	157 ± 5	34 ± 7
1000	9 ± 2		11 ± 2		27 ± 5	164 ± 14	18 ± 4
2500	12 ± 5		12 ± 5		18 ± 1	99 ± 7	8 ± 2
5000	8 ± 1 p		5 ± 1 p		11 ± 2 p	36 ± 10 p	2 ± 1 p
Positive Control	2-AA		2-AA		2-AA	2-AA	2-AA
Dose (µg/plate)	2.5		2.5		2.5	2.5	10
Number of revertant colonies/plate	219 ± 34		173 ± 24		4806 ± 849	4719 ± 196	346 ± 48
NaN3: sodium azide 2AA: 2-Aminoanthracene 4-NOPD: 4-nitro-o-phenylene-diamine MMS: methyl methane silfonate				p: Precipitate

Table 2: Results of Experiment 2 (pre-incubation).

	Number of revertant colonies (mean of 3 plates ± SD)
Without S9
Test substance (µg/plate)	TA 1535	TA 1537			TA 98	TA 100		WP2 uvr A
DMSO	9 ± 2	8 ± 3			26 ± 8	149 ± 9		44 ± 8
Untreated	10 ± 3	13 ± 3			22 ± 9	189 ± 5		42 ± 10
10		9 ± 3			29 ± 8			44 ± 8
33	11 ± 2	9 ± 4			25 ± 3	143 ± 12		40 ± 3
100	9 ± 3	10 ± 2			25 ± 4	151 ± 6		42 ± 1
333	8 ± 2	9 ± 3			26 ± 5	126 ± 17		25 ± 4
1000	13 ± 3	8 ± 3			21 ± 6	114 ± 26		14 ± 5
2500	10 ± 2	11 ± 3 r			20 ± 3	75 ± 17		16 ± 1
5000	10 ± 1 r	9 ± 6 r			18 ± 3	48 ± 23 r		17 ± 8
Positive Control	NaN3	4-NOPD			4-NOPD	NaN3		MMS
Dose (µg/plate)	10	50			10	10		2
Number of revertant colonies/plate	1225 ± 59	89 ± 10			462 ± 22	1884 ± 169		732 ± 13
With S9
Test substance (µg/plate)	TA 1535		TA 1537		TA 98	TA 100	WP2 uvr A
DMSO	13 ± 2		11 ± 5		32 ± 4	136 ± 7	43 ± 10
Untreated	10 ± 6		8 ± 3		44 ± 9	206 ± 10	59 ± 5
10			13 ± 2		39 ± 7		52 ± 7
33	9 ± 2		11 ± 5		35 ± 7	130 ± 6	49 ± 8
100	13 ± 3		8 ± 2		30 ± 6	153 ± 21	42 ± 12
333	10 ± 3		11 ± 3		33 ± 6	145 ± 14	36 ± 4
1000	10 ± 4		10 ± 4		39 ± 10 r	85 ± 1 r	18 ± 4
2500	5 ± 2 r, m		5 ± 2 r, m		9 ± 1 r, m	5 ± 1 r, m	18 ± 4
5000	2 ± 1 r, m		7 ± 4 r, m		6 ± 2 r, m	5 ± 1 r, m	8 ± 2
Positive Control	2-AA		2-AA		2-AA	2-AA	2-AA
Dose (µg/plate)	2.5		2.5		2.5	2.5	10
Number of revertant colonies/plate	369 ± 24		160 ± 7		4201 ± 214	2905 ± 543	377 ± 14
NaN3: sodium azide 2AA: 2-Aminoanthracene 4-NOPD: 4-nitro-o-phenylene-diamine MMS: methyl methane silfonate				r: reduced background growth m: manual count

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five strains (TA 1535, TA 1537, TA 98, TA 100 and WP2 uvrA) tested with and without metabolic activation up to 5000 µg/plate.

Executive summary:

Ames test according to OECD 471 and GLP was performed. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was 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: Strains TA 1535 and TA 100: 33; 100; 333; 1000; 2500; and 5000 µg/plate; the remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate.

No substantial increase in revertant colony numbers of any of the five tester strains was observed any dose level, neither in the presence nor absence of metabolic activation (S9 mix). In conclusion, the test item did not induce gene mutations by base pair

changes or frameshifts in the genome of the strains used.