3,4,5-trifluorophenol

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:

July 26, 2000 - January 10, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

29.Dec.1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

September 11, 1989

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3,4,5-trifluorophenol

EC Number:

436-120-9

EC Name:

3,4,5-trifluorophenol

Cas Number:

99627-05-1

Molecular formula:

C6H3F3O

IUPAC Name:

3,4,5-trifluorophenol

Method

Target gene:

All these strains contain mutations in the histidine operon, thus imposing a requirement for histidine in the growth medium.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 5.00, 15.8, 50.0, 158 and 500 µg per plate

The test material concentrations used were selected according to the EEC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test item and positive controls N-Ethyl-N'-nitro-N-nitrosoguanidine, cumene hydroperoxide, 2-Aminoanthracene, Benzo[a] pyrene) and Ethanol for positive control 9-Aminoanthracene

- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents. Analysis of the historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason only the respective solvent control was used as the negative control in this study.

- Justification for percentage of solvent in the final culture medium: Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of water (added as the solvent) will dilute the top agar, usually the maximum amount of solvent is limited to 100 µl per plate for water and 10 µL per plate for DMSO, ethanol, acetone or other organic solvents. Only the highest test material concentration may be plated with either 316 µL water or 31.6 µL organic solvent.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

without S9 mix

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.
- Number of independent experiments : 2

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Incubation of plates was performed at + 37 °C for 2 to 3 days.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate in the 1st test series.

STUDY RESULTS :
- Signs of toxicity :
toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.
- Mean number of revertant colonies per plate and standard deviation : see tables 1-4

Any other information on results incl. tables

Table 1: Results / Series No.: 1

Positive Controls: Individual and Mean Number of Revertant Colonies         

Strain	Positive Control	Concentr. [µg/plate]	+ /- S9-Mix #	Mean revertant colonies per plate	SD*	Individual revertant colonies per plate
TA 98	DAUN	2	-	643	196	869	538	521
TA 100	ENNG	5	-	904	41	866	947	899
TA 102	CUM	67	-	396	37	421	414	354
TA 1535	ENNG	10	-	332	12	329	321	345
TA 1537	9-AA	50	-	1683	331	1727	1990	1333
WP2	ENNG	5	-	1327	83	1264	1296	1421
TA 98	2-AA	1	+	322	19	338	301	326
TA 100	2-AA	1	+	537	25	564	532	514
TA 102	B (a) p	10	+	806	3	804	805	809
TA 1535	2-AA	1	+	151	17	152	134	167
TA 1537	2-AA	2	+	92	34	95	124	56
WP2	2-AA	10	+	1459	IB	1477	1441	1459

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 2: Results/ Series No.: 2

Positive Controls: Individual and Mean Number of Revertant Colonies             

Strain	Positive Control	Concentr. [µg/plate]	+ /- S9-Mix #	Mean revertant colonies per plate	SD*	Individual revertant colonies per plate
TA 98	DAUN	2	-	93	18	73	97	108
TA 100	ENNG	5	-	520	39	565	500	494
TA 102	CUM	150	-	661	72	703	702	578
TA 1535	ENNG	10	-	187	11	194	192	174
TA 1537	9-AA	50	-	186	40	144	191	224
WP2	ENNG	5	-	1103	12	1117	1096	1097
TA 98	2-AA	10	+	329	150	434	396	158
TA 100	2-AA	10	+	333	30	302	336	361
TA 102	B (a) p	10	+	775	120	790	887	649
TA 1535	2-AA	2	+	94	11	107	87	649
TA 1537	2-AA	20	+	42	10	17	49	30
WP2	2-AA	10	+	933	81	861	1020	918

 

#        Test material incubated in the presence

(+)       or absence

(-)        of S9-Mix

*          Standard deviation

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

 

Table 3: Results / Series No.: 1

Summary of the Mean Number of Revertant Colonies              

Test material	Concentr. [µg/plate]	+ /- S9-Mix	Mean revertant colonies / plate
			TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2
Solvent control Test item		-	14	97	269	12	4	128
	5	-	14	91	264	12	3	119
	15.8	-	17	96	268	12	6	127
	50	-	15	99	249	15	4	122
	158	-	16	112	242	12	7	114
	500 PE	-	8	73	189	11	2	53
	1580 PE	-	0	3	2	2	0	2
	5000 PE	-	0	3	0	2	0	2
Solvent control Test item		+	20	120	271	13	7	170
	5	+	25	121	277	20	9	164
	15.8	+	27	132	288	14	8	180
	50	+	26	142	278	13	9	167
	158	+	27	143	342	17	8	130
	500 PE	+	22	130	214	12	5	81
	1580 PE	+	2	4	2	3	0	3
	5000 PE	+	0	5	0	3	0	5
Postive controls	Name	-	DAUN	ENNG	CUM	ENNG	9-AA	ENNG
	Conc (µg/plate)		2	5	67	10	50	5
	Revert. / plate		343	904	396	332	1683	1327
	Name	+	2-AA	2-AA	B (a) p	2-AA	2-AA	2-AA
	Conc (µg/plate)		1	1	10	1	2	10
	Revert. / plate		322	537	806	151	92	1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

2-AA    2-Aminoanthracene

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

Table 4 Results / Series No.: 2

Summary of the Mean Number of Revertant Colonies              

Test material	Concentr. [µg/plate]	+ /- S9-Mix	Mean revertant colonies / plate
			TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2
Solvent control Test item		-	15	93	218	11	5	130
	5	-	13	87	219	7	7	130
	15.8	-	12	92	220	11	8	127
	50	-	13	88	222	12	4	122
	158	-	16	88	221	15	7	119
	500	-	11	79	206	11	6	91
Solvent control Test item		+	25	140	275	18	6	134
	5	+	22	139	254	17	5	133
	15.8	+	20	153	226	20	7	151
	50	+	20	156	259	16	3	144
	158	+	26	176	266	18	6	128
	500	+	30	153	261	12	6	115
Postive controls	Name	-	DAUN	ENNG	CUM	ENNG	9-AA	ENNG
	Conc (µg/plate)		2	5	150	10	50	5
	Revert. / plate		93	520	661	187	186	1327
	Name	+	B (a) p	B (a) p	B (a) p	2-AA	B (a) p	2-AA
	Conc (µg/plate)		10	10	10	2	20	10
	Revert. / plate		329	333	775	94	42	1459

 

DAUN  Daunomycin

ENNG  N-Ethyl-N’-nitro-N-nitroso-guanidine

CUM    Cumene hydroperoxide

B (a) p Benzo(a)pyrene

9-AA    9-Aminoacridine

2-AA    2-Aminoanthracene