4-fluoroaniline

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-05-05 to 1999-09-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

EPA 712-C-98-221, August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5-difluoroaniline

EC Number:

206-752-8

EC Name:

3,5-difluoroaniline

Cas Number:

372-39-4

Molecular formula:

C6H5F2N

IUPAC Name:

3,5-difluoroaniline

Test material form:

other: solid

Method

Species / strain

Species / strain / cell type:

lymphocytes: CeII Line L5178Y

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640-HAT and nomral RPMI 1640 medium to reduce the amount of spontaneous mutants
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix - Phenobarbital i.p. and β-Naphthoflavone induced

Test concentrations with justification for top dose:

Experiment I (with S9 mix, 4 h): 20.3, 40.6, 81.3, 162.5, 325.0 µg/mL
Experiment I (without S9 mix, 4 h); Experiment II (without S9 mix, 24 h): 40.6, 81.3, 162.5, 325.0, 650.0 µg/mL
Experiment II (with S9 mix, 4 h): 25.0, 50.0, 100.0, 200.0, 400.0 µg/mL

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
The final concentration of DMSO in the culture medium did not exceed 0.5 % (v/v).

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

dissolved in DMSO

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Remarks:

MMS: 13 µg/mL; 3-MC 3 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium

DURATION
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 72 hours

After the expression period the cultures were seeded into microtiter plates.
Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4x 10^3 cells in selective medium (see below) with TFT (SERVA, D-69042 Heidelberg).
The cloning efficiency (viability) was determined by seeding about 2.5 cells per well into microtiter plates (same medium without TFT). The plates are incubated at 37°C in 4.5 % CO2 / 95.5 % humidified air for 10 - 15 days to determine the plating efficiency and to evaluate mutagenicity. Then the plates were evaluated manually.

Culture medium: RPMI 1640 medium supplemented with 15 % horse serum (HS), 100 U/100 µg/mL Penicilin/ Streptomycin, 220 µg/mL Sodium-Pyruvate, and 1.25 U/mL Amphotericin used as antifungal.

Cloning Medium:
RPMI 1640 (complete culture medium)

Selective Medium:
RPMI 1640 (complete culture medium) by addition of 5 µg/m TFT.

NUMBER OF CELLS EVALUATED: 1 x 10E7

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth
Test article concentrations between 10.2 and 1300 µg/mL (with and without S9-mix) were used to evaluate toxicity.

Evaluation criteria:

A test substance is classified as positive if it induces either a reproducible concentration related increase in the mutant frequency or a reproducible positive response for at least one of the test points.

A test substance producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A significant response is described as follows:
The test substance is classified as mutagenic if it reproducibly induces with at least one of the concentrations a mutation frequency that is two times higher than the mean spontaneous mutation frequency in the experiment.

The test substance is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such evaluation may be considered independently of an enhancement factor for induced mutants.

However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.

Results of test groups are rejected if either, the relative total growth or relative suspension growth is less than 10 % of the solvent control or the cloning efficiency II after the expression period is less than 20 %.

Statistics:

No statistical evaluation is required.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
After 4 hours of treatment strong toxic effects were observed at 325.0 µg/mL and above in the absence and at 81.3 µg/mL and above in the presence of S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The highest negative control value (127 colonies) and the highest solvent control value (121 colonies) exceeded the range of our historical control values slightly. This effect was considered as irrelevant since the corresponding solvent or negative control was well within the range of the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I, strong toxic effects occurred at 650 µg/mL in the absence and 81.3 µg/mL and above in both parallel cultures in the presence of metabolic activation (4 h treatment).
Strong toxic effects were observed in the second experiment at 650 µg/mL without metabolic activation (continuous treatment for 24 h) and at 25 µg/mL and above in both parallel cultures with metabolic activation (4 h treatment).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of experiment I

			Experiment I, culture I				Experiment I, culture II
	Per ml	mix	Relative cloning efficiency I	Relative total growth	Mutant colonies/ 108 Cells	Induction factor	Relative cloning efficiency I	Relative total growth	Mutant colonies/ 108 Cells	Induction factor
Column	1	2	3	4	5	6	7	8	9	10
Neg. control with medium		-	100.0	100.0	29		100.0	100.0	43
Neg. control with DMSO		-	100.0	100.0	50	1.0	100.0	100.0	70	1.0
Pos. control with MMS	13.0	-	53.2	82.0	124	2.5	92.8	84.9	193	2.7
Test substance	20.3	-	101.5	Culture was not continued			74.5	Culture was not continued
Test substance	40.6	-	97.1	131.5	48	1.0	84.3	127.0	55	0.8
Test substance	81.3	-	109.5	93.7	52	1.0	94.8	90.5	50	0.8
Test substance	162.5	-	133.2	88.7	46	0.9	107.9	90.3	73	1.0
Test substance	325.0	-	82.8	39.3	71	1.4	103.8	53.4	56	0.8
Test substance	650.0	-	41.4	20.0	60	1.2	47.7	24.5	61	0.9
Neg. control with medium		+	100.0	100.0	72		100.0	100.0	86
Neg. control with DMSO		+	100.0	100.0	71	1.0	100.0	100.0	47	1.0
Pos. control with 3-MC	3.0	+	96.9	68.5	221	3.1	118.7	81.2	199	4.2
Test substance	20.3	+	61.8	43.5	63	0.9	64.9	51.3	47	1.0
Test substance	40.6	+	49.8	44.5	91	1.3	74.1	29.0	72	1.5
Test substance	81.3	+	32.1	22.0	110	1.6	40.0	26.2	91	1.9
Test substance	162.5	+	34.3	14.8	132	1.9	35.6	22.7	69	1.5
Test substance	325.0	+	16.0	13.9	93	1.3	20.3	7.5	141	3.0
Test substance	650.0	+	1.7	Culture was not continued			2.5	Culture was not continued

   

 

 

Summary of experiment II

 

			Experiment II, culture I				Experiment II, culture II
	Per ml	mix	Relative cloning efficiency I	Relative total growth	Mutant colonies/ 108 Cells	Induction factor	Relative cloning efficiency I	Relative total growth	Mutant colonies/ 108 Cells	Induction factor
Column	1	2	3	4	5	6	7	8	9	10
Neg. control with medium		-	100.0	100.0	80		100.0	100.0	91
Neg. control with DMSO		-	100.0	100.0	54	1.0	100.0	100.0	64	1.0
Pos. control with MMS	13.0	-	28.2	33.6	604	11.1	47.4	36.4	690	10.8
Test substance	20.3	-	74.2	Culture was not continued			133.5	Culture was not continued
Test substance	40.6	-	66.1	51.0	106	1.9	101.9	60.8	120	1.9
Test substance	81.3	-	60.5	69.7	69	1.3	91.3	134.1	44	0.7
Test substance	162.5	-	26.5	68.5	65	1.2	83.9	88.9	65	1.0
Test substance	325.0	-	43.1	34.1	69	1.3	65.6	75.6	72	1.1
Test substance	650.0	-	13.9	14.0	133	2.4	32.2	30.8	113	1.8
Neg. control with medium		+	100.0	100.0	112		100.0	100.0	127
Neg. control with DMSO		+	100.0	100.0	121	1.0	100.0	100.0	103	1.0
Pos. control with 3-MC	3.0	+	57.6	84.4	283	2.3	86.7	77.2	280	2.7
Test substance	12.5	+	44.0	Culture was not continued			32.5	Culture was not continued
Test substance	25.0	+	27.0	35.4	117	1.0	28.8	21.4	147	1.4
Test substance	50.0	+	28.9	18.3	187	1.6	24.6	12.3	155	1.5
Test substance	100.0	+	9.0	8.7	330	2.7	14.0	7.3	219	2.1
Test substance	200.0	+	12.2	5.2	344	2.9	6.4	5.2	174	1.7
Test substance	400.0	+	1.3	0.8	438	3.6	0.9	0.3	311	3.0

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative