Acetoacetanilide

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed OECD and GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acetoacetanilide

EC Number:

202-996-4

EC Name:

Acetoacetanilide

Cas Number:

102-01-2

Molecular formula:

C10H11NO2

IUPAC Name:

3-oxo-N-phenylbutanamide

Details on test material:

- Name of test material (as cited in study report): Acetessiganilid TTR

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I and II with WP2 uvrA: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment I with
the Salmonella strains: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II with
the Salmonella strains: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test and pre-incubation assay


DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

 Summary of Results Pre-Experiment/Experiment I with WP2 uvrA

Study Name: 1343800	Study Code: Harlan CCR 1343800
Experiment: 1343800 VV Plate	Date Plated: 07/06/2010
Assay Conditions:	Date Counted: 10/06/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				WP2 uvrA
Without Activation	DMSO			50 ± 8
	Untreated			60 ± 9
	Acetessiganilid TTR	3 µg		46 ± 7
		10 µg		48 ± 2
		33 µg		56 ± 11
		100 µg		68 ± 8
		333 µg		64 ± 7
		1000 µg		66 ± 11
		2500 µg		61 ± 3
		5000 µg		42 ± 1^P
	MMS	3.0 µL		1174 ± 29
With Activation	DMSO			68 ± 2
	Untreated			71 ± 7
	Acetessiganilid TTR	3 µg		67 ± 7
		10 µg		65 ± 10
		33 µg		69 ± 5
		100 µg		75 ± 2
		333 µg		94 ± 12
		1000 µg		92 ± 10
		2500 µg		84 ± 7^P
		5000 µg		51 ± 6^P
	2-AA	10.0 µg		470 ± 23

Key to Positive Controls		Key to Plate Postfix Codes
MMS 2-AA	methyl methane sulfonate 2-aminoanthracene	P	Precipitate

   Summary of Results Pre-Experiment/Experiment II with WP2 uvrA

Study Name: 1343800	Study Code: Harlan CCR 1343800
Experiment: 1343800 HV2 Pre	Date Plated: 07/06/2010
Assay Conditions:	Date Counted: 10/06/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				WP2 uvrA
Without Activation	DMSO			56 ± 9
	Untreated			58 ± 3
	Acetessiganilid TTR	3 µg		52 ± 4
		10 µg		57 ± 8
		33 µg		63 ± 5
		100 µg		64 ± 6
		333 µg		64 ± 3
		1000 µg		72 ± 6
		2500 µg		63 ± 4
		5000 µg		36 ± 6
	MMS	3.0 µL		661 ± 27
With Activation	DMSO			74 ± 4
	Untreated			64 ± 3
	Acetessiganilid TTR	3 µg		66 ± 8
		10 µg		71 ± 15
		33 µg		84 ± 5
		100 µg		93 ± 14
		333 µg		97 ± 6
		1000 µg		111 ± 6
		2500 µg		79 ± 18^P
		5000 µg		54 ± 4^P
	2-AA	10.0 µg		343 ± 29

Key to Positive Controls		Key to Plate Postfix Codes
MMS 2-AA	methyl methane sulfonate 2-aminoanthracene	P	Precipitate

 Summary of Results Experiment I with the Salmonella strains

Study Name: 1343800	Study Code: Harlan CCR 1343800
Experiment: 1343800 HV1 Plate	Date Plated: 15/06/2010
Assay Conditions:	Date Counted: 18/06/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100
Without Activation	DMSO			13 ± 4	10 ± 1	20 ± 4	136 ± 4
	Untreated			16 ± 5	8 ± 1	29 ± 3	208 ± 109
	Acetessiganilid	3 µg		14 ± 2	11 ± 3	32 ± 5	147 ± 19
	TTR	10 µg		14 ± 3	7 ± 1	23 ± 3	136 ± 15
		33 µg		13 ± 5	9 ± 1	18 ± 3	146 ± 17
		100 µg		13 ± 3	10 ± 3	21 ± 4	157 ± 10
		333 µg		17 ± 4	7 ± 2	25 ± 2	175 ± 7
		1000 µg		17 ± 5	5 ± 1	20 ± 1	165 ± 21
		2500 µg		15 ± 1	8 ± 2	21 ± 2	166 ± 9
		5000 µg		14 ± 2	7 ± 2	23 ± 7	163 ± 14
	NaN3	10 µg		1798 ± 93			2015 ± 94
	4-NOPD	10 µg				539 ± 61
	4-NOPD	50 µg			379 ± 43
With Activation	DMSO			20 ± 1	22 ± 7	32 ± 5	184 ± 14
	Untreated			19 ± 3	18 ± 2	32 ± 4	201 ± 23
	Acetessiganilid	3 µg		19 ± 6	20 ± 4	32 ± 3	169 ± 1
	TTR	10 µg		15 ± 5	16 ± 4	36 ± 10	181 ± 22
		33 µg		18 ± 6	17 ± 5	41 ± 6	183 ± 12
		100 µg		18 ± 5	25 ± 8	43 ± 12	173 ± 7
		333 µg		23 ± 2	23 ± 5	36 ± 3	180 ± 3
		1000 µg		21 ± 1	19 ± 9	31 ± 5	185 ± 15
		2500 µg		20 ± 5	24 ± 5	34 ± 3	188 ± 22
		5000 µg		23 ± 2	26 ± 2	33 ± 5	174 ± 14
	2-AA	2.5 µg		432 ± 86	684 ± 43	3275 ± 835	4378 ± 274

Key to Positive Controls
NaN3 2-AA 4-NOPD	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine

 Summary of Results Experiment II with the Salmonella strains

Study Name: 1343800	Study Code: Harlan CCR 1343800
Experiment: 1343800 HV2a Pre	Date Plated: 22/06/2010
Assay Conditions:	Date Counted: 29/06/2010

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100
Without Activation	DMSO			13 ± 3	15 ± 5	22 ± 1	102 ± 6
	Untreated			15 ± 6	14 ± 5	30 ± 6	121 ± 8
	Acetessiganilid	33 µg		15 ± 3	14 ± 1	22 ± 5	106 ± 2
	TTR	100 µg		14 ± 1	14 ± 1	22 ± 7	118 ± 7
		333 µg		14 ± 2	13 ± 3	17 ± 4	116 ± 11
		1000 µg		13 ± 6	9 ± 1	19 ± 4	119 ± 12
		2500 µg		12 ± 1	11 ± 4	22 ± 5	122 ± 16
		5000 µg		14 ± 2	15 ± 7	18 ± 2	133 ± 3
	NaN3	10 µg		1198 ± 50			1242 ± 16
	4-NOPD	10 µg				407 ± 39
	4-NOPD	50 µg			98 ± 9
With Activation	DMSO			21 ± 3	19 ± 5	40 ± 9	131 ± 15
	Untreated			14 ± 3	19 ± 3	55 ± 6	133 ± 25
	Acetessiganilid	33 µg		22 ± 8	17 ± 3	39 ± 3	124 ± 8
	TTR	100 µg		23 ± 5	20 ± 1	39 ± 11	144 ± 15
		333 µg		19 ± 3	20 ± 3	31 ± 2	144 ± 15
		1000 µg		19 ± 5	17 ± 6	28 ± 5	131 ± 16
		2500 µg		16 ± 3	12 ± 2	30 ± 4	139 ± 15
		5000 µg		17 ± 2	16 ± 3	30 ± 7	123 ± 13
	2-AA	2.5 µg		248 ± 39	331 ± 30	1082 ± 73	1851 ± 19

Key to Positive Controls
NaN3 2-AA 4-NOPD	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

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, Acetessiganilid TTR is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

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

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Experiment I and II with WP2 uvrA:   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment I with
the Salmonella strains:                      3; 10;33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II with
the Salmonella strains:                      33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal back­ground growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabol­ic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Acetessiganilid TTR 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.

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

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, Acetessiganilid TTR is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.