4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

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:

2010-11-17 to 2010-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

EC Number:

638-734-4

Cas Number:

157248-24-3

Molecular formula:

C₁₇H₁₈F₂O

IUPAC Name:

4-ethoxy-2,3-difluoro-4'-propyl-1,1'-biphenyl

Test material form:

solid

Method

Target gene:

Salmonella typhimurium TA98, TA100, TA102, TA1535, TA1537: mutations in the histidine operon
Escherichia coli WP2 uvrA: defect in one of the genes for tryptophan biosynthesis

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: his G 46, uvrB, rfa

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: his C 3076, uvrB, rfa

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: his D 3052, uvrB, rfa + R-factor

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: his G 46, uvrB, rfa + R-factor

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: his G 428, rfa + R-factor

Species / strain 6

Species / strain / cell type:

E. coli WP2 uvr A

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9 fraction derived from rat liver homogenate; from male Wistar rats, HSdCpb:Wu (Harlan Winkelmann, Borchen, Germany), age 6-8 weeks, pretreated with Aroclor (500 mg/kg bw)
- method of preparation of S9 mix: Please refer to “Any other information on materials”.
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 30 % S9 in the S9 mix were used in the first and second test series, respectively.
- quality controls of S9: Each S9 batch was tested for its metabolic activity using specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for each S9 batch.

Test concentrations with justification for top dose:

1st series: 5, 15.8, 50, 158, 500, 1580, 5000 µg/plate
2nd series: 50, 88.9, 158, 281 and 500 µg/plate

5000 µg/plate was chosen as the appropriate maximum test material concentration. The test material concentrations used were selected according to the EEC, OECD and Japanese guidelines for this test system.

Vehicle / solvent:

- Solvent used: acetone

- Justification for choice of solvent: Test item was sufficiently soluble in the solvent. Analysis of the historical data of the laboratory and experience of other research groups showed that the amounts of the selected solvent used has no influence on the number of spontaneous revertants of any strain.

- Justification for percentage of solvent in the final culture medium: not specified

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cumene hydroperoxide

other: daunomycin (DAUN)

Remarks:

without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- 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: 2-3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Increase of revertant colonies

Rationale for test conditions:

Test conditions in line with respective test guidelines were used.

Evaluation criteria:

The criteria for assessment of colony numbers, based upon the historical controls of the laboratory and statistical considerations, were established (please refer to “Any other information on materials and methods”. All further results, ranging between "no" and "clear", are assessed as "weak increases”.

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 and the results of each series be discussed case by case.

Statistics:

Not specified

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

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 100

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 3

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 4

Key result

Species / strain:

S. typhimurium TA 1537

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 5

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

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolality: not specified
- Possibility of evaporation from medium: no
- Water solubility: Sufficiently soluble in selected solvent.
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations ≥ 500 µg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data

Ames test:
- Signs of toxicity: No toxicity was observed.
- Individual plate counts: Please refer to attached document under "Overall remarks, attachments".
- Mean number of revertant colonies per plate and standard deviation: Please refer to “Any other information on results".

HISTORICAL CONTROL DATA
Not specified

Any other information on results incl. tables

Summary 1st series

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2 uvrA
Without Activation	Acetonene			19 ± 6	112 ± 11	260 ± 24	32 ± 5	13 ± 3	20 ± 4
	Test material	5.00		20 ± 8	121 ± 15	248 ± 11	32 ± 6	17 ± 5	26 ± 6
		15.8		19 ± 10	116 ± 7	271 ± 17	36 ± 6	15 ± 2	24 ± 7
		50.0		17 ± 8	106 ± 15	311 ± 15	30 ± 2	15 ± 8	28 ± 3
		158		16 ± 4	113 ± 4	328 ± 21	30 ± 5	15 ± 5	28 ± 10
		500		18 ± 6^E	112 ± 12^E	332 ± 11^E	26 ± 4^E	14 ± 4^E	25 ± 6^E
		1580		19 ± 3^E	110 ± 17^E	270 ± 42^E	26 ± 5^E	9 ± 1^E	26 ± 7^E
		5000		20 ± 3^E	108 ± 5^E	268 ± 8^E	23 ± 5^E	10 ± 5^E	25 ± 5^E
	DAUN	1.00		241 ± 35
	NaN3	2.00			746 ± 68		452 ± 32
	CUM	200				1717 ± 101
	9-AA	50.0						618 ± 86
	NQO	2.00							1275 ± 14
With Activation	Acetone			29 ± 8	118 ± 13	345 ± 25	24 ± 4	14 ± 4	34 ± 4
	Test material	5.00		38 ± 5	110 ± 18	351 ± 20	23 ± 4	20 ± 4	33 ± 12
		15.8		28 ± 7	102 ± 20	380 ± 59	20 ± 9	17 ± 6	30 ± 4
		50.0		28 ± 5	108 ± 17	391 ± 48	25 ± 7	20 ± 4	37 ± 6
		158		35 ± 8	136 ± 8	440 ± 23	22 ± 4	19 ± 4	33 ± 5
		500		27 ± 2^E	130 ± 5^E	433 ± 26^E	18 ± 6^E	19 ± 4^E	38 ± 4^E
		1580		29 ± 4^E	114 ± 11^E	412 ± 31^E	17 ± 2^E	15 ± 4^E	29 ± 4^E
		5000		23 ± 6^E	99 ± 9^E	397 ± 20^E	18 ± 5^E	10 ± 2^E	36 ± 4^E
	2-AA	2.00		601 ± 35	546 ± 158		134 ± 11
	2-AA	5.00						279 ± 26
	2-AA	10.0							463 ± 38
	B(a)p	10.0				2454 ± 62

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA CUM B(a)p 9-AA DAUN NQO	Sodium azide 2-Aminoanthracene Cumene hydroperoxide Benzo[a]pyrene 9-Aminoacridine Daunomycin 4-Nitroquinoline-N-oxide	E	Precipitation until end of experiment

Summary 2nd series

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 102	TA 1535	TA 1537	WP2 uvrA
Without Activation	Acetone			21 ± 5	140 ± 13	308 ± 28	28 ± 5	13 ± 7	29 ± 10
	Test material	50.0		22 ± 3	137 ± 15	367 ± 15	28 ± 3	13 ± 8	33 ± 3
		88.9		26 ± 9	137 ± 21	335 ± 14	29 ± 3	8 ± 4	32 ± 5
		158		22 ± 2	127 ± 9	383 ± 36	37 ± 14	14 ± 1	30 ± 7
		281		20 ± 9	126 ± 12	367 ± 30	29 ± 8	13 ± 4	31 ± 4
		500		28 ± 2^E	127 ± 10^E	388 ± 55^E	40 ± 2^E	19 ± 6^E	37 ± 2^E
	DAUN	1.00		253 ± 17
	NaN3	2.00			843 ± 88		735 ± 33
	CUM	200				1543 ± 103
	9-AA	50.0						702 ± 158
	NQO	2.00							1151 ± 104
With Activation	Acetone			30 ± 5	96 ± 8	293 ± 10	20 ± 4	17 ± 4	37 ± 5
	Test material	50.0		26 ± 2	107 ± 10	373 ± 20	19 ± 3	18 ± 6	38 ± 12
		88.9		37 ± 9	109 ± 8	398 ± 24	22 ± 7	16 ± 3	39 ± 5
		158		29 ± 8	115 ± 17	437 ± 32	23 ± 2	19 ± 6	29 ± 5
		281		37 ± 8	110 ± 7	420 ± 47	22 ± 8	18 ± 8	38 ± 10
		500		35 ± 1^E	108 ± 24^E	425 ± 35^E	21 ± 3^E	18 ± 8^E	36 ± 11^E
	2-AA	2.00		225 ± 34			101 ± 17
	2-AA	5.00			477 ± 100
	2-AA	10.0						190 ± 18	296 ± 27
	B(a)p	10.0				797 ± 104

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA CUM B(a)p 9-AA DAUN NQO	Sodium azide 2-Aminoanthracene Cumene hydroperoxide Benzo[a]pyrene 9-Aminoacridine Daunomycin 4-Nitroquinoline-N-oxide	E	Precipitation until end of experiment

Applicant's summary and conclusion

Conclusions:

The test item was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation test with and without metabolic activation.

Executive summary:

The investigations for the mutagenic potential of the test material were performed according to OECD 471 using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively. The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred in the concentration range between 500 and 5000 µg/plate. Toxicity to the bacteria was not observed. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions.