3',4',5'-trifluorobiphenyl-2-amine

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): 3',4',5'-Trifluoro-biphenyl-2-ylamine
- Physical state: Solid, dusky pink up to red
- Analytical purity: 99.67%

Method

Target gene:

his / trp

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 fraction / hamster liver S9 mix (reductive S9 mix)

Test concentrations with justification for top dose:

see below

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Good solubility

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 30 min
- Exposure duration: 48 - 72 h


NUMBER OF REPLICATIONS: 3 plates / dose / experiment

DETERMINATION OF CYTOTOXICITY
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer
is recorded for all test groups both with and without S9 mix in all experiments.

EXPERIMENTS:

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 20; 100; 500; 2 500 and 5 000 μg/plate
Type of test: Ames standard plate test with and without rat liver S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 25; 50; 100; 200 and 400 μg/plate (Salmonella strains)
0; 156.3; 312.5; 625; 1 250 and 2 500 μg/plate (E. coli WP2 uvrA)
Type of test: Ames standard plate test with and without rat liver S9 mix
Number of plates: 3 test plates per dose or per control

3rd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 4; 20; 100; 200 and 400 μg/plate (Salmonella strains)
0; 20; 100; 500; 2 500 and 5 000 μg/plate (E. coli WP2 uvrA)
Type of test: Prival preincubation test with and without hamster liver S9 mix
Number of plates: 3 test plates per dose or per control

POSITIVE CONTROLS

The following positive controls are used to check the mutability of the bacteria and the activity
of the S9 mix:
With rat liver S9 mix
• 2-aminoanthracene (2-AA) (SIGMA, A-1381)1)
- 2.5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO
- strain: Escherichia coli WP2 uvrA
With hamster liver S9 mix
• 2-aminoanthracene (2-AA) (SIGMA, A-1381)1)
- 10 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
• Congo red (SIGMA, C-6277)1)
- 210 μg/plate, dissolved in DMSO
- strain: TA 98
• benzidine (SIGMA, B-1883)1)
- 55 μg/plate, dissolved in DMSO
- strain: TA 98
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (FLUKA, 68051)1)
- 5 μg/plate, dissolved in DMSO
- strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (SIGMA, N-9504)1)
- 10 μg/plate, dissolved in DMSO
- strain: TA 98
• 9-aminoacridine (AAC) (SIGMA, A-1135)1)
- 100 μg/plate, dissolved in DMSO
- strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141)1)
- 5 μg/plate, dissolved in DMSO
- strain: E. coli WP2 uvrA
The stability of the selected positive controls is well-defined under the selected culture
conditions, since they are well-established reference mutagens.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Key result

Species / strain:

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

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:

Cytotoxicity

A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed in the Ames standard plate test from about 100 μg/plate onward in the absence and presence of induced rat liver S9 mix.
In the Prival preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed from 200 μg/plate onward in the absence and presence of uninduced hamster liver S9 mix.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (Ames standard plate test and Prival preincubation assay).

The slight increase in the number of revertants observed with TA 100 in the 1st Experiment in the presence of metabolic activation with induced rat liver S9 mix was not corroborated in the 2nd Experiment. Therefore, this observation has to be regarded as biologically irrelevant. Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.