Benzenamine, 4-fluoro-N-(1-methylethyl)-

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:

06 Jul - 27 Aug 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Fifth strain (E. coli WP2 or S. typhimurium TA102) is missing. However, based on the results of the present study the test substance is considered mutagenic. Additional testing with a fifth strain (WP2uvrA or TA 102) will not provide relevant information for the hazard and risk assessment of the substance. Therefore, this study is considered sufficient for hazard assessment purposes of the test substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted in 1983

Deviations:

yes

Remarks:

Fifth strain (E. coli WP2 or S. typhimurium TA102) is missing. The present study was conducted in accordance with the previous version of the OECD TG 471. This deviation does not affect the final outcome of the study, nor will further testing.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-100-7

EC Name:

-

Cas Number:

70441-63-3

Molecular formula:

C9H12FN

IUPAC Name:

4-fluoro-N-(propan-2-yl)aniline

Test material form:

liquid

Method

Target gene:

his operon

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 rats treated with Aroclor 1254.

Test concentrations with justification for top dose:

Concentration range of the first experiment (with and without metabolic activation): 8, 40, 200, 1000, and 5000 µg/plate, all strains
Concentration range of the second experiment (with and without metabolic activation): 60, 120, 240, 480, and 960 µg/plate , all strains
Concentration range of the third experiment (with metabolic activation): 400, 600, 800, 1000, 1200, and 1400 µg/plate, TA 1537 and TA 98
Concentration range of the fourth experiment (with and without metabolic activation): 60, 120, 240, 480, and 960 µg/plate, TA 100

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: Nitrofurantoin (NF, 0.2 µg/plate, -S9, TA 100); 4-Nitro-1,2-phenylene diamine (4-NPDA, 10 µg/plate, -S9, TA1537); 4-Nitro-1,2-phenylene diamine (4-NPDA, 0.5 µg/plate, -S9, TA98); 2-aminoanthracene (2-AA, 3 µg/plate, +S9, all strains, S9 activity control)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 4
- Number of independent experiments: 4

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation).

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): The count was made after the plates had been incubated for 48 hours at 37 °C.

METHODS FOR MEASUREMENT OF CYTOTOXICITY :
- Methods: 1) Background growth inhibition; 2) Toxic effect of the test substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative control; 3) The titer was determined. Total bacterial counts were taken on two plates for each tested concentration.

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
Colony count using automatic counter.

Evaluation criteria:

The following criteria determined the acceptance of an assay:
1) The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and the laboratories' own historical data.
2) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
3) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points 1) - 3) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Assessment 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. However, these guidelines may be overruled by good scientific judgement.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Statistics:

Mean values and standard deviation were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥960 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥960 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥960 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥960 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥1400 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

≥200 µg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥1400 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA
- Positive historical control data: The mean values of the positive controls of the present experiment were within the laboratories' historical control data range.
- Negative (solvent/vehicle) historical control data: The mean values of the DMSO vehicle control of the present experiment were within the laboratories' historical control data range.

Remarks on result:

other: In the present study, the test item showed bacterial mutagenicity in the Salmonella typhimurium strain TA 98 in presence of S9 mix; allover the test substance was highly cytotoxic in all strains.

Any other information on results incl. tables

Table 1. Ames Test: results of experiment 1 (plate incorporation)

With (+) or without (-) S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	mean of 4 plates ± SD
		Base-pair substitution type		Frameshift type
		TA 1535	TA 100	TA 1537	TA 98
–	DMSO	9 ± 3	68 ± 8	11 ± 1	23 ± 9
–	8	5 ± 2	73 ± 7	8 ± 4	31 ± 16
–	40	8 ± 2	71 ± 3	8 ± 3	21 ± 5
–	200	11 ± 4	76 ± 6	8 ± 3	18 ± 2
–	1000	10 ± 4	82 ± 17	11 ± 5	21 ± 5
–	5000	b	b	b	b
Positive controls, -S9	Name	Na-azide	NF	4-NPDA	4-NPDA
	Concentrations (μg/plate)	10	0.2	10	0.5
	Mean No. of colonies/plate (average of 4 ± SD)	496 ± 35*	240 ± 23*	51 ± 4*	49 ± 7*
+	DMSO	8 ± 2	78 ± 8	5 ± 2	27 ± 10
+	8	6 ± 1	80 ± 10	9 ± 3	32 ± 10
+	40	9 ± 2	75 ± 12	7 ± 2	28 ± 8
+	200	9 ± 3	78 ± 11	9 ± 3	111 ± 18*
+	1000	9 ± 6	132 ± 28	17 ± 3	277 ± 61*
+	5000	b	b	b	b
Positive control, +S9	Name	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	3	3	3	3
	Mean No. of colonies/plate (average of 4 ± SD)	85 ± 6*	550 ± 150*	217 ± 40*	1467 ± 88*

SD = standard deviation

Na-azide = sodium azide

NF = nitrofurantoin

4-NPDA = 4-nitro-1,2-phenylenediamine

2-AA = 2-aminoanthracene

b = bacteriotoxic effect

* = mutagenic effect

Table 2. Ames Test: results of experiment 2 (plate incorporation)

With (+) or without (-) S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	mean of 4 plates ± SD
		Base-pair substitution type		Frameshift type
		TA 1535	TA 100^1	TA 1537	TA 98
–	DMSO	10 ± 4		13 ± 2	22 ± 4
–	60	10 ± 3		8 ± 2	21 ± 4
–	120	12 ± 4		8 ± 1	21 ± 6
–	240	14 ± 5		8 ± 2	27 ± 3
–	480	10 ± 5		6 ± 3	28 ± 7
–	960	10 ± 3		10 ± 3	29 ± 8
Positive controls, -S9	Name	Na-azide		4-NPDA	4-NPDA
	Concentrations (μg/plate)	10		10	0.5
	Mean No. of colonies/plate (average of 4 ± SD)	475 ± 45*		48 ± 16*	56 ± 18*
+	DMSO	15 ± 3		14 ± 3	34 ± 7
+	60	10 ± 4		13 ± 4	42 ± 10
+	120	13 ± 5		10 ± 2	51 ± 9
+	240	10 ± 1		10 ± 2	56 ± 9*
+	480	15 ± 2		10 ± 4	79 ± 10*
+	960	15 ± 2		11 ± 4	81 ± 11*
Positive control, +S9	Name	2-AA		2-AA	2-AA
	Concentrations (μg/plate)	3		3	3
	Mean No. of colonies/plate (average of 4 ± SD)	188 ± 36*		109 ± 13*	1012 ± 90*

SD = standard deviation

1 = not used for assessment beacause of low vehicle control counts

Na-azide = Sodium azide

4-NPDA = 4-Nitro-1,2-phenylenediamine

2-AA = 2-Aminoanthracene

* = mutagenic effect

Table 3. Ames Test: results of experiment 3 (plate incorporation)

With (+) S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	mean of 4 plates ± SD
		Frameshift type
		TA 1537	TA 98
+	DMSO	6 ± 4	18 ± 2
+	400	12 ± 5	47 ± 12*
+	600	9 ± 3	70 ± 20*
+	800	10 ± 3	64 ± 9*
+	1000	11 ± 6	49 ± 13*
+	1200	12 ± 2	78 ± 11*
+	1400	12 ± 4	95 ± 12*
Positive control, +S9	Name	2-AA	2-AA
	Concentrations (μg/plate)	3	3
	Mean No. of colonies/plate (average of 4 ± SD)	191 ± 42*	648 ± 44*

SD = standard deviation

2-AA = 2-Aminoanthracene

* = mutagenic effect

Table 4. Ames Test: results of experiment 4 (plate incorporation)

With (+) or without (-) S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	mean of 4 plates ± SD
		Base-pair substitution type
		TA 100
–	DMSO	95 ± 16
–	60	107 ± 23
–	120	104 ± 16
–	240	104 ± 14
–	480	114 ± 1
–	960	132 ± 13
Positive controls, -S9	Name	NF
	Concentrations (μg/plate)	0.2
	Mean No. of colonies/plate (average of 4 ± SD)	262 ± 40*
+	DMSO	144 ± 20
+	60	139 ± 11
+	120	142 ± 15
+	240	175 ± 21
+	480	196 ± 10
+	960	202 ± 18
Positive control, +S9	Name	2-AA
	Concentrations (μg/plate)	3
	Mean No. of colonies/plate (average of 4 ± SD)	1017 ± 76*

SD = standard deviation

NF = nitrofurantoin

2-AA = 2-aminoanthracene

* = mutagenic effect

Applicant's summary and conclusion

Conclusions:

Under the conditions of the present Ames test conducted with 4 strains of Salmonella typhimurium (TA 1535, TA 100, TA 1537, and TA 98), a clear positive result indicating mutagenicity is reported for S. typhimurium TA 98, in the presence of metabolic activation.