Phenethyl isobutyrate

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:

2001-03-16 to 2001-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Phenethyl isobutyrate

EC Number:

203-116-1

EC Name:

Phenethyl isobutyrate

Cas Number:

103-48-0

Molecular formula:

C12H16O2

IUPAC Name:

2-phenylethyl 2-methylpropanoate

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

experiment 1: 0, 50, 150, 500, 1500, 5000 µg/plate
experiment 2: 0, 5, 15, 50, 150, 500, 1500 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: with S9 mix: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The number of spontaneous revertants observed using each of the five strains was close to those previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

Statistics:

The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each
dosage level was calculated using the X2-test (Mohn and Ellenberger, 1977).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the absence of S9 mix, bacteriotoxicity was observed at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was not observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9-mix the substance was not bacteriotoxic.

Any other information on results incl. tables

Table 1: Number of revertants per plate (mean of three plates), Experiment 1 and 2, without metabolic activation

	strain TA 98		strain TA 100		strain TA 102		strain TA 1535		strain TA 1537
conc. [µg]	Exp1	Exp2	Exp1	Exp2	Exp1	Exp2	Exp1	Exp2	Exp1	Exp2
control	35	31	83	99	316	278	22	10	13	11
solvent control	32	31	88	90	265	257	24	10	11	11
5	33		87			183	26		8
15	34	20	79	79	191	165	27	14	9	13
50	39	30	88	78	249	214	29	14	11	13
150	35	28	82	85	239	236	12	11	9	9
500	28	26	86	84	118T	143T	20	11	7	9
1500		15T		47T	85T			7T		4T
Sodium azide 0.7 µg			528	499			841	471
2-nitrofluorene 2.5 µg	549	598
9-aminoacridine 50µg									115	180
MitomycinC 0.15 µg					807	881

T: Cytotoxicity

Table 2: Number of revertants per plate (mean of three plates), Experiment 1 and 2, with metabolic activation

	strain TA 98		strain TA 100		strain TA 102		strain TA 1535		strain TA 1537
conc. [µg]	Exp1	Exp2	Exp1	Exp2	Exp1	Exp2	Exp1	Exp2	Exp1	Exp2
control	27	26	89	90	289	320	12	11	17	10
solvent control	21	23	80	89	275	299	13	13	14	14
50	23	28	82	89	272	256	7	12	11	15
150	23	23	73	89	265	278	8	13	12	13
500	22	22	75	84	256	259	11	15	10	13
1500	20	23	74	82	235	273	15	15	9	15
5000	17	23	72	82	223	228	13	14	14	14
2-aminoanthracene 0.8 µg/plate	799	1018	917	753
2-aminoanthracene 0.9 µg/plate					538		308	91
2-aminoanthracene 1.0 µg/plate						592
2-aminoanthracene 1.7 µg/plate									114	203

T: Cytotoxicity

Applicant's summary and conclusion

Conclusions:

The test substance was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The test substance was tested for its mutagenicity in the bacterial reverse mutation assay. Therefore, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 were used. The test was performed using the standard plate incorporation assay with and without S9 -mix (Aroclor 1254 induced). Two independet experiments were carried out using concentrations of 50 -5000 µg and 5 -1500 µg/plate. In the presence of S9-mix no bacteriotoxicity was observed. In the absence of S9-mix the test substance was bacteriotoxic towards the strain TA102 at 500 µg/plate and towards the strains TA1535, TA1537, TA98, and TA100 at 1500 µg/plate. As mutagenicity controls, sodium azide, 2 -nitrofluorene, 9 -aminoacridine and mitomycin C were used in the absence of S9 -mix and 2 -aminoanthracene was used in the in the presence of S9 mix. As a result of this assay, mutagenicity was not observed.