2,2'-[(3-acetamidophenyl)imino]diethyl diacetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Salmonella Mutagenicity Tests: V. Results from the Testing of 311 Chemicals

Author:

Errol Zeiger, Beth Anderson, Steve Haworth, Timothy Lawlor, and Kristien Mortelmans

Year:

1992

Bibliographic source:

Environmental and Molecular Mutagenesis Volume 19, Supplement 21 :2-141 (1992)

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to evaluate the mutagenic nature of ethyl acetate

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl acetate

EC Number:

205-500-4

EC Name:

Ethyl acetate

Cas Number:

141-78-6

Molecular formula:

C4H8O2

IUPAC Name:

ethyl acetate

Test material form:

liquid

Details on test material:

- Name of test material: Ethyl acetate
- IUPAC name: Ethyl acetate
- Molecular formula: C4H8O2
- Molecular weight: 88.1052 g/mol
- Substance type: Organic
- Physical State: Liquid

Specific details on test material used for the study:

- Name of test material: Ethyl acetate
- IUPAC name: Ethyl acetate
- Molecular formula: C4H8O2
- Molecular weight: 88.1052 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: Vendor’s purity: 99.5% Analyzed purity: 99%
- Impurities (identity and concentrations): No data

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA100, TA1535, TA1537, TA97, TA98

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The S-9 (9,000 x g supernatant) fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers

Test concentrations with justification for top dose:

0, 100, 333, 1000, 3333 or 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (TA98 and TA1538; Without S9); 2-aminoanthracene (With S9; all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: Plates were machine counted unless precipitate was present which interfered with the count, or the color of the test chemical on the plate reduced the contrast between the colonies and the agar.

Rationale for test conditions:

No data

Evaluation criteria:

The plates were observed for a dose dependent increase in the number of Histidine- independent (his+) colonies.

Evaluations were made at both the individual trial and chemical levels.

Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or nonmutagenic (-), depending on the magnitude of the increase in his+ revertants, and the shape of the dose response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “+ W”, if only a single dose was elevated over the control, or if a weak increase was not dose-related. The distinctions between a questionable response and a nonmutagenic or weakly mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a trial to be judged mutagenic.

A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537, TA97, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: All chemicals were run initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100. Toxic concentrations were defined as those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Any other information on results incl. tables

Table: Results for the test chemical Ethyl acetate

Dose (µg/plate)	TA100
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	152	0.6	154	9.9	171	9.9	145	2.0	128	1.2
100	135	5.4	151	0.3	173	3.8	151	11.7	122	10.2
300	143	8.3	151	7.2	169	4.4	160	7.6	150	7.1
1000	135	4.9	127	12.2	181	11.9	151	15.1	166	3.2
3333	116	3.5	129	11.1	180	9.8	124	4.5	140	9.6
10000	124	7.3	116	4.6	131	20.5	119	5.7	121	9.1
Positive control	412	8.4	835	37.2	545	10.8	602	17.8	842	33.8

 

Dose (µg/plate)	TA1535
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	20	1.0	11	2.2	14	2.4	9	1.7	15	0.7
100	16	0.7	11	2.2	12	1.8	13	0.6	12	1.0
300	23	3.2	9	0.6	12	2.0	9	1.7	10	2.3
1000	20	2.3	10	0.9	12	3.0	10	1.9	9	2.1
3333	17	2.6	7	0.9	10	0.9	8	0.6	15	2.6
10000	19	5.2	8	0.6	10	0.3	8	0.0	16	1.9
Positive control	562	16.7	255	22.5	403	13.1	168	13.3	96	2.6

 

Dose (µg/plate)	TA1537
	-S9		10% HLI		30% HLI
	Mean	SEM	Mean	SEM	Mean	SEM
0	8	1.3	10	1.9	8	0.7
100	6	2.0	12	1.6	9	0.9
300	10	2.1	15	1.2	10	0.9
1000	9	2.1	11	2.7	11	1.7
3333	9	0.6	13	1.3	9	1.7
10000	7	0.6	10	1.0	9	0.7
Positive control	330	20.6	26	2.0	37	1.5

 

 

Dose (µg/plate)	TA97
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	149	1.9	180	14.2	191	17.1	166	4.2	216	4.7
100	169	4.5	179	5.2	200	7.7	196	29.8	222	5.0
300	167	1.5	179	12.9	198	12.1	197	9.6	233	1.7
1000	138	3.4	175	15.3	185	8.7	208	0.6	208	2.9
3333	153	2.9	163	12.8	179	17.5	188	11.8	203	8.8
10000	149	6.3	161	5.8	177	5.8	191	11.1	190	24.3
Positive control	1158	52.6	634	44.5	330	13.1	480	9.2	404	1.7

 

Dose (µg/plate)	TA98
	-S9		10% HLI		30% HLI		10% RLI		30% RLI
	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM	Mean	SEM
0	20	0.3	36	3.8	34	1.3	30	3.2	48	1.7
100	17	0.3	30	1.7	37	4.0	29	1.8	37	2.1
300	13	2.6	29	2.1	35	4.7	25	3.7	31	1.2
1000	15	0.3	33	3.6	29	5.0	23	2.8	38	3.2
3333	17	3.5	5	2.3	34	1.9	23	2.3	35	2.9
10000	19	1.0	34	4.0	41	4.8	27	3.3	40	4.6
Positive control	643	23.8	786	32.0	283	13.6	435	11.0	134	5.9

 

Applicant's summary and conclusion

Conclusions:

Ethyl acetate did not induce mutation in Salmonella typhimurium TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of ethyl acetate. The study was performed using Salmonella typhimurium strains TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO as solvent and used at dose levels 0, 100, 333, 1000, 3333 or 10000 µg/plate by the preincubation method. The doses were selected on the basis of preliminary dose range finding study and concurrent solvent and positive controls were included in the study. Ethyl acetatedid not induce mutation in Salmonella typhimurium TA97, TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 metabolic activation system and hence is not likely to classify as a gene mutant in vitro.