4'-methoxyacetophenone

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:

2017-05-02 to 2017-06-14

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 July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 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'-methoxyacetophenone

EC Number:

202-815-9

EC Name:

4'-methoxyacetophenone

Cas Number:

100-06-1

Molecular formula:

C9H10O2

IUPAC Name:

1-(4-methoxyphenyl)ethanone

Method

Target gene:

Salmonella typhimurium: histidine

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:

Liver homogenate (S9) from Phenobarbital/ß-naphtoflavone pretreated rats

Test concentrations with justification for top dose:

In a pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. Since toxic effects were observed six or seven concentrations were tested in the main experiment. 5000 μg/plate, respectively 2500 μg/plate were chosen as maximal concentration. The following concentrations were tested in the final test:
Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

With and without (vehicle control) DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

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

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Pre-experiment for toxicity: In agar (plate incorporation)
- Experiment I: In agar (plate incorporation)
- Experiment II: Preincubation
- Selective Agar: Plates with selective agar (without histidine/tryptophan) were used

DURATION
- Preincubation period: Mix of 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension incubated at 37 °C for 60 minutes
- Exposure duration: 48 hours at 37 °C in the dark after solidification of the plates

NUMBER OF REPLICATIONS:
- two independent experiments with three replications

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies

Evaluation criteria:

A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeded 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 was observed. A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration. An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory. Therefore, no statistics were performed.

Results and discussion

Test results

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA

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
No precipitation of the test item occurred up to the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment I: Bacteriotoxic towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix.

- Experiment II: Bacteriotoxic towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate.Bacteriotoxic towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix.

Any other information on results incl. tables

Table 1: Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		8 ± 3	13 ± 2	28 ± 7	156 ± 7	44 ± 2
	Untreated		13 ± 2	12 ± 2	28 ± 8	170 ± 22	36 ± 8
	Test Item	3 µg	7 ± 1	9 ± 3	20 ± 6	159 ± 29	37 ± 2
		10 µg	6 ± 1	11 ± 2	24 ± 9	165 ± 13	40 ± 11
		33 µg	10 ± 1	13 ± 3	24 ± 5	171 ± 12	46 ± 10
		100 µg	17 ± 3	13 ± 3	28 ± 8	167 ± 15	44 ± 7
		333 µg	7 ± 3	11 ± 4	24 ± 8	166 ± 22	35 ± 9
		1000 µg	8 ± 1	15 ± 4	18 ± 3	171 ± 14	27 ± 3
		2500 µg	8 ± 2	15 ± 1	22 ± 4	129 ± 13	11 ± 1
		5000 µg	9 ± 3	8 ± 2	13 ± 4	57 ± 16	3 ± 1
	NaN3	10 µg	1120 ± 231			1993 ± 36
	4-NOPD	10 µg			300 ± 32
	4-NOPD	50 µg		65 ± 9
	MMS	2.0 µL					996 ± 11
With Activation	DMSO		10 ± 2	16 ± 6	28 ± 9	158 ± 24	49 ± 18
	Untreated		9 ± 2	16 ± 7	41 ± 5	148 ± 10	53 ± 10
	Test Item	3 µg	10 ± 4	15 ± 1	38 ± 3	149 ± 7	51 ± 12
		10 µg	13 ± 3	15 ± 1	29 ± 8	142 ± 11	62 ± 8
		33 µg	8 ± 2	16 ± 4	22 ± 2	137 ± 6	40 ± 2
		100 µg	9 ± 2	16 ± 4	29 ± 3	144 ± 19	54 ± 8
		333 µg	10 ± 3	15 ± 6	34 ± 10	134 ± 15	41 ± 8
		1000 µg	12 ± 0	27 ± 2	28 ± 8	147 ± 3	31 ± 9
		2500 µg	11 ± 1	20 ± 6	27 ± 9	130 ± 15	17 ± 4
		5000 µg	7 ± 4	12 ± 3	21 ± 1	75 ± 7	8 ± 3
	2-AA	2.5 µg	441 ± 19	212 ± 36	3255 ± 284	4212 ± 229
	2-AA	10 µg					296 ± 14

Key to Positive Controls:

NaN3: sodium azide

2-AA: 2 -aminoanthracene

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

MMS: methyl methane sulfonate

Table 2: Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		10 ± 3	10 ± 1	27 ± 10	120 ± 10	41 ± 7
	Untreated		11 ± 2	9 ± 09 ± 0	37 ± 8	213 ± 3	31 ± 10
	Test Item	3 µg					38 ± 8
		10 µg					38 ± 8
		33 µg	11 ± 5	8 ± 2	29 ± 8	106 ± 14	36 ± 9
		100 µg	13 ± 4	8 ± 2	23 ± 4	139 ± 3	32 ± 4
		333 µg	11 ± 4	9 ± 4	33 ± 12	119 ± 3	31 ± 5
		1000 µg	12 ± 3	10 ± 4	24 ± 3	96 ± 4	22 ± 3
		2500 µg	13 ± 3	6 ± 1	24 ± 4	67 ± 12	17 ± 6
		5000 µg	9 ± 3	4 ± 0	18 ± 5	0 ± 1
	NaN3	10 µg	1413 ± 31			2123 ± 55
	4-NOPD	10 µg			354 ± 7
	4-NOPD	50 µg		115 ± 21
	MMS	2.0 µL					636 ± 43
With Activation	DMSO		14 ± 1	11 ± 2	30 ± 5	125 ± 11	45 ± 9
	Untreated		12 ± 1	16 ± 7	48 ± 5	174 ± 11	50 ± 17
	Test Item	3 µg					55 ± 5
		10 µg					46 ± 2
		33 µg	12 ± 2	10 ± 2	36 ± 10	113 ± 12	54 ± 8
		100 µg	13 ± 2	10 ± 4	36 ± 12	131 ± 7	48 ± 13
		333 µg	13 ± 5	11 ± 3	32 ± 2	139 ± 15	37 ± 6
		1000 µg	11 ± 1	16 ± 1	38 ± 11	83 ± 8	35 ± 4
		2500 µg	10 ± 1	15 ± 3	31 ± 9	37 ± 5	19 ± 2
		5000 µg	7 ± 0	7 ± 1	6 ± 0	3 ± 1
	2-AA	2.5 µg	424 ± 18	110 ± 14	4237 ± 917	3262 ± 234
	2-AA	10 µg					453 ± 29

Key to Positive Controls:

NaN3: sodium azide

2-AA: 2 -aminoanthracene

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

MMS: methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic to Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.

Executive summary:

An in vitro reverse mutation assay study (Ames) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3μg/plate and 5000μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed in Experiment I towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix. In Experiment II: the test substance showed bacteriotoxic effects towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate as well as towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix. No substantial increase in revertant colony numbers of any of the tester strains was detected following treatment with the test substance at any dose 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. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.