Acetamide, N-(4-fluorophenyl)-2-hydroxy-N-(1-methylethyl)-

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

Study period:

May - July 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

incomplete strain selection (tester strain with AT reversion site missing)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted version 1983

Deviations:

no

Remarks:

no prior stability testing of compound, compared to current guideline only 4 strains tested and incomplete strain selection (tester strain with AT reversion site missing)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-(4-fluorophenyl)-2-hydroxy-N-(propan-2-yl)acetamide

EC Number:

611-084-9

Cas Number:

54041-17-7

Molecular formula:

C11H14FN

IUPAC Name:

N-(4-fluorophenyl)-2-hydroxy-N-(propan-2-yl)acetamide

Specific details on test material used for the study:

Batch/Lot No.: 17001/93
Manufacturer: Bayer AG
Identity analysis: May 10, 1993
Stability: until March 29, 1995

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male SD rats treated with aroclor 1254 (metabolic activity of each batch was verified with reference mutagen(s)

Test concentrations with justification for top dose:

0, 8, 40, 200, 1000 and 5000 µg/plate (tested up to the recommended maximum dose)

Vehicle / solvent:

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

sodium azide

other: nitrofurantoin (NF), 4-nitro-1,2-phenylene diamine (4-NPDA), 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); in suspension (preincubation method)

DURATION
- Preincubation period:
20 min
- Exposure duration:
48 h

NUMBER OF REPLICATIONS:
4 plates/strain, 2 replications

DETERMINATION OF CYTOTOXICITY
- Method: The first method was a gross appraisal of background growth on the plates for mutant determination. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

Rationale for test conditions:

The results of the first experiment were considered as a pre-test for toxicity. Since solubility was not limited, 5000 µg/plate was chosen as the highest dose. Doses of repeats were chosen on the basis of the results obtained in the first experiment.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative.

The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/or the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

Statistics:

Means of colony numbers were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

slight cytotoxicity at 5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight cytotoxicity at 5000 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 3: Results as mean values without S9 mix

Compound	Method	concentration	TA 1535	TA 100	TA 1537	TA 98
DMSO	plate	0	11	70	9	21
Test substance		8	10	87	8	24
		40	13	79	8	26
		200	8	88	8	19
		1000	14	70	8	23
		5000	13	74	3	20
Sodium azide			825	-	-	-
NF			-	337	-	-
4-NPDA			-	-	64	74
DMSO	tube	0	10	97	7	23
Test substance		8	10	85	9	22
		40	10	98	7	26
		200	7	88	9	28
		1000	9	93	7	28
		5000	8	73	4	24
Sodium azide			564	-	-	-
NF			-	424	-	-
4-NPDA			-	-	58	70

NF: nitrofurantoin, 4-NPDA : 4-nitro-1,2-phenylene diamine

 

 

Table 3: Table 2: Results as mean values with S9 mix

Compound	Method	concentration	TA 1535	TA 100	TA 1537	TA 98
DMSO	plate	0	14	108	7	33
Test substance		8	14	128	8	33
		40	11	120	9	34
		200	11	113	9	25
		1000	11	124	9	23
		5000	14	108	7	30
2-AA			59	775	56	1286
DMSO	tube	0	11	129	10	34
Test substance		8	12	80	1	37
		40	13	91	09	28
		200	10	64	9	28
		1000	12	68	5	33
		5000	8	64	4	30
2-AA			111	903	231	1102

2-AA: 2-aminoanthracene

 

Applicant's summary and conclusion

Conclusions:

FOE 5043-Hydroxy was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Executive summary:

FOE 5043-Hydroxy was investigated in an independent repeat using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 ng per tube after preincubation for 20 minutes at 37°C on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses up to and including 40 ng per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had only a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes. Evidence of mutagenic activity of FOE 5043-Hydroxy was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.