Ethanone, 1-(4-methoxyphenyl)-2-[(3-methoxyphenyl)thio]-

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:

July to Sept 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

469-300-0

EC Name:

-

Cas Number:

63675-73-0

Molecular formula:

Hill Empirical Formula: C16H16O3S CAS Empirical Formula: C16H16O3S

IUPAC Name:

1-(4-methoxyphenyl)-2-[(3-methoxyphenyl)sulfanyl]ethan-1-one

Test material form:

solid: particulate/powder

Details on test material:

Lot no: 151105Purity - 101.39%

Method

Target gene:

Histidine locus in several strains of Salmonella typhimurium and the typtophan locus of E coli strain(WP2uvrA)

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 exogenous mammalian activation system

Test concentrations with justification for top dose:

Concentration range in the Preliminary rangefinding mutagenicity assay (with & without metabolic activation): > 33.3 ... < 5000 µg/plate. Note: in the presence of S9 mix, decreases in the mean number of revertants per plate were observed with the Salmonella tester strains only at 5000mcg of Beta Ketosulfide per plate.The results of the preliminary rangefinding mutagenicity assay were used to select the doses tested in the confirmatory mutagenicity assay....all performed in the presence and absence of S9 mix at 33.1, 100, 333, 1000, 2500 and 5000mcg of Beta Ketosulfide per plate.

Vehicle / solvent:

Solvent: Dimethylsulfoxide (DMSO)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: ICR-191, 2 aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation method NUMBER OF REPLICATIONS: Three of each replicate was incubated for 48 hours at 37C DETERMINATION OF CYTOTOXICITY: Cytotoxicity was indicated by the presence of "pinpoint" colonies and the absence of lawn background.

Rationale for test conditions:

The bacterial reverse mutation assay detects point mutations, both frameshifts and/or base pair s ubstitutions. The strains of Salmonella typhimurium and Escherichia coli used in this assay are histidine and tryptopban auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his-) or tryptophan (lrp-) dependent cells are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his+) or tryptophan (rrp+) inde pendence are able to form colonies. The trace amount of histidine or tryptophan in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ or trp+ revertants are readily discernable as colonies against the limited background growth of the his- or trp- cells. By utilizing several different tester strains, base pair substitution mutations and frameshift mutations can be detected. The bacterial reverse mutation assay has been shown to be a sensitive, rapid, and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes.

Evaluation criteria:

VALID STUDY CRITERIAThe vehicle control (Dimethyl Sulfoxide) must show a normal range of bacterial colonies for each bacterial strain and should be consistent with historical data. The positive controls N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), 2 -nitrofluorene (2NF), 9 -ami noacridine (9AmAc), and 2 -aminoanthracene (2AA) must show a mutagenic response and should be consistent with historical data. In the absence of toxicity or precipitation, a maximum treatment concentration of 5000 mcg/plate is sufficiently high to support study validity.POSITIVE RESPONSE CRITERIATest substance is judged to have induced a positive response when a concentration -related increase in revertants is observed in which the number of revertants exceeds the control values by at least 2 - fold (strains TA98, TA100, and WP2uvrA) or at least 3 -fold (strains TA1535 or TA1537) for at least t wo successive test article concentrations. When the above criteria are met for only one concentration, the determination of a positive response will be made on the basis of scientific judgment relative to the quality of the concentration response finding.NEGATIVE RESPONSE CRITERIAA test article is considered to produce a negative response when the criteria for a positive response is not met and the conditions for a valid assay have been satisfied.

Statistics:

No statistical analysis was used

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle 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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

For the confirmatory mutagenicity assay, all data was acceptable and no positive increases in the me an number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.

Any other information on results incl. tables

Table1: Mean and Std Deviation from Triplicate plates - An evaluation of compound 202723 to S. ty[himurium and E.coli

Treatment	µg/plate	TA98	TA100	TA1535	TA1537	WP2uvrA	Background Lawn^a
Revertant Colony Counts With Activation
DMSOa	50 mcl	19  ± 7	120  ± 6	11  ± 4	6  ± 0	15  ± 3	N
	33.3 mcg	22   ± 12	124   ± 16	11  ± 4	6  ±4	15  ± 3	N
	100 mcg	15  ±3	122  ±9	11  ± 5	7  ±1	17  ±3	N
	333 mcg	21  ±5	111  ±9	14  ±3	4  ±4	10  ±2	N
	1000 mcg	18  ±4	114  ±11	8  ±3	5  ±6	11  ±4	N
	2500 mcg	9  ±2	94  ±6	8  ±1	5  ±1	13  ±1	NP
	5000 mcg	13  ±4	77  ±4	6  ±1	2  ±1	13  ±6	NP
Positive control ^b		311  ±17	713  ±58	111  ±23	99  ±11	157  ±68	N
		Revertant Colony Counts without Activation
DMSO	50mcl	13  ±4	86  ±4	6  ±2	3  ±1	11  ±3	N
	33.3mcg	14  ±2	82  ±8	12  ±8	6  ±3	12  ±7	N
	100mcg	13  ±3	82 ±12	4 ±2	7 ±4	16 ±4	N
	333mcg	8 ±2	69 ±12	8 ±2	3 ±2	13 ±2	N
	1000mcg	14 ±2	82 ±9	8 ±2	6 ±2	14 ±2	N
	2500mcg	13 ±6	76 ±9	9 ±2	6 ±4	9 ±2	NP
	5000mcg	14 ±3	75 ±14	9 ±2	6 ±1	15 ±1	NP
Positive control ^c		290 ±54	990 ±74	644 ±50	389 ±38	265 ±35	N

^a Background Lawn Evaluation codes:

N=normal, R=reduced, O=obscured, A=absent, Precipitate

^b TA98 = benzo[a]pyrene 2.5mcg/plate

TA100 = 2 -aminoanthracene 2.5mcg/place

TA1535 = 2 -aminoanthracene 2.5mcg/place

TA1537 = 2 -aminoanthracene 2.5mcg/place

WP2uvrA = 2 -aminoanthracene 25.0mcg/place

c TA98 = 2 -nitrofluorene 1.0 mcg/plate

TA100 = sodium azide 2.0mcg/plate

TA1535= sodium azide 2.0mcg/plate

TA1537 = ICR-191 2.0mcg/plate

WP2uvrA = 4 -nitroquinoline-N-oxide 1.0mcg/plate

Applicant's summary and conclusion

Conclusions:

The results of the Salmonella typhimurium-Escherichia coli / Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that under the conditions of this study, Beta Ketosulfide did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor TM induced rat liver (S9).

Executive summary:

The objective of this study was to evaluate Beta Ketosulfide (155766)for the ability to induce reverse mutations at the histidine locus in Salmonella typhimurium tester strains TA98,TA100, TA1535,and TA1537,and at the tryptophan locus of Escherichia coli tester strain WP2uvrA.  Evaluations were conducted in the presence or absence of an exogenous mammalian activation system (S9) containing microsomal enzymes.

The preliminary range finding mutagenicity assay was performed using all tester strains in both the presence and absence of S9 mix at 1.60, 5.00, 16.0,50.0, 160, 500,1600,and 5000 mcg of Beta Ketosulfide per plate along with the appropriate vehicle and positive controls. All doses of test article, vehicle controls and positive controls were plated in duplicate.

The confirmatory mutagenicity assay was performed using all tester strains in both the presence and absence of S9 mix at 33.3, 100, 333, 1000, 2500,and 5000mcg of Beta Ketosulfide per plate.  All doses of test article, vehicle controls and positive controls were plated in triplicate.

The results of the Salmonella typhimurium- Escherichia coli /Mammalian-Microsome Reverse Mutation Assay with a Confirmatory Assay indicate that, under the conditions of this study, BetaKetosulfide(155766)did not cause a positive increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes prepared from Aroclor™-induced rat liver (S9). The responses to the positive controls demonstrated that the test system was sensitive to chemical mutagens.