trans-1-[4-(4-Propylcyclohexyl)-1-cyclohexen-1-yl]-4- (trifluoromethyl)-benzene

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:

Feb 16 - Apr 20, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines for this test system

Version / remarks:

1993

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

trans-4-propyl-4''-(trifluoromethyl)-1,1',2,2',3,3',4,5,6,6'-decahydro[1,1':4',1''-terphenyl]

EC Number:

847-991-3

Cas Number:

90278-32-3

Molecular formula:

C₂₂H₂₉F₃

IUPAC Name:

trans-4-propyl-4''-(trifluoromethyl)-1,1',2,2',3,3',4,5,6,6'-decahydro[1,1':4',1''-terphenyl]

Test material form:

solid: crystalline

Method

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor (pKM101)

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain 5

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

trp-, uvrA

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from beta-Naphthoflavone/Phenobarbital-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the OECD and Japanese guidelines for this test system. The test material showed best solubility in Acetone and 5000 µg/plate was chosen as the appropriate maximum concentration due to limited solubility.
1. Series: 0.281, 0.889, 2.81, 8.89, 28.1, 88.9, 281 µg/plate
2. Series: 0.889, 2.81, 8.89, 28.1, 88.9 µg/plate

Vehicle / solvent:

Acetone

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

sodium azide

other: Daunomycin

Remarks:

without S9

Controls 2

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

Details on test system and experimental conditions:

Bacterial strains were tested in accordance with the plate incorporation method. 3 parallel plates were used for each concentration step of the test material. The incubation of plates was performed at 36-38°C for 2 days. Liver S9 mix from rats pre-treated with beta-naphthoflavone/phenobarbital was used as the metabolic activation system. Two experimental series were performed, containing 10% S9 ind the 1st and 20% S9 in the 2nd series.

Rationale for test conditions:

according to Guideline

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
• the assay is considered valid and
• a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
• an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
• a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
• the assay is considered valid and
• none of the above-mentioned criteria are met

Whenever colony counts remain within the historical range of negative controls, such increases are considered as biologically not meaningful. In general, two series of experiments must be performed.

Results which only partially satisfied the above criteria were dealt with on a case by case basis. Biological relevance was considered, for example consistency of response within and between concentrations and (where applicable) between experiments.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Results of 1^stseries

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	Acetone			36 ± 10	130 ± 10	21 ± 7	9 ± 4	30 ± 11
	Test material	0.281		30 ± 10	111 ± 4	23 ± 6	7 ± 1	29 ± 4
		0.889		38 ± 1	117 ± 6	22 ± 5	9 ± 4	31 ± 8
		2.81		29 ± 5	121 ± 8	19 ± 4	9 ± 6	29 ± 7
		8.89		28 ± 7	125 ± 1	19 ± 3	7 ± 1	31 ± 4
		28.1		33 ± 5 E	118 ± 9 E	18 ± 5 E	10 ± 4 E	30 ± 7 E
		88.9		31 ± 2 E	117 ± 6 E	18 ± 2 E	7 ± 3 E	33 ± 4 E
		281		28 ± 3 E	134 ± 13 E	16 ± 4 E	4 ± 2 E	27 ± 5 E
	DAUN	2.00		872 ± 129
	NaN3	2.00			1480 ± 102	753 ± 55
	9-AA	50.0					1317 ± 190
	NQO	2.00						1879 ± 138
With Activation	Acetone			41 ± 4	114 ± 11	19 ± 4	8 ± 2	31 ± 9
	Test item	0.281		34 ± 2	125 ± 13	18 ± 7	8 ± 6	32 ± 11
		0.889		33 ± 4	117 ± 6	19 ± 7	4 ± 1	34 ± 9
		2.81		34 ± 10	129 ± 17	15 ± 2	6 ± 3	41 ± 8
		8.89		41 ± 11	121 ± 19	19 ± 3	7 ± 5	33 ± 5
		28.1		49 ± 15 E	116 ± 12 E	29 ± 7 E	9 ± 1 E	42 ± 10 E
		88.9		42 ± 10 E	121 ± 6 E	17 ± 6 E	6 ± 3 E	40 ± 10 E
		281		33 ± 10 E	134 ± 5 E	19 ± 1 E	6 ± 2 E	33 ± 4 E
	2-AA	2.00		923 ± 68	1773 ± 46
	2-AA	5.00				121 ± 22	300 ± 42
	2-AA	10.0						328 ± 65

Results of 2^ndseries

Metabolic Activation	Test Material	Concentr. [µg/plate]		Revertants per plate (Mean ± SD)
				TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	Acetone			32 ± 5	133 ± 9	29 ± 4	9 ± 4	33 ± 4
	Test item	0.889		34 ± 7	129 ± 9	44 ± 2	10 ± 4	27 ± 3
		2.81		35 ± 8	131 ± 9	36 ± 9	8 ± 5	25 ± 4
		8.89		33 ± 8	135 ± 5	36 ± 6	10 ± 7	31 ± 9
		28.1		41 ± 7	150 ± 9	30 ± 7	14 ± 2	28 ± 6
		88.9		34 ± 6 E	140 ± 10 E	30 ± 3 E	5 ± 4 E	29 ± 1 E
	DAUN	2.00		736 ± 22
	NaN3	2.00			1468 ± 82	881 ± 56
	9-AA	50.0					1443 ± 125
	NQO	2.00						1234 ± 95
With Activation	Acetone			43 ± 5	131 ± 10	22 ± 8	14 ± 3	36 ± 7
	Test item	0.889		37 ± 8	120 ± 10	25 ± 2	10 ± 3	26 ± 5
		2.81		40 ± 8	113 ± 3	22 ± 2	11 ± 3	40 ± 6
		8.89		37 ± 12	134 ± 16	25 ± 3	14 ± 3	38 ± 12
		28.1		42 ± 15	135 ± 6	28 ± 7	16 ± 11	34 ± 7
		88.9		43 ± 2 E	124 ± 6 E	21 ± 4 E	13 ± 5 E	36 ± 2 E
	2-AA	2.00		1287 ± 97	1804 ± 144
	2-AA	5.00				146 ± 28	341 ± 35
	2-AA	10.0						489 ± 34

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 9-AA DAUN NQO	Sodium azide 2-Aminoanthracene 9-Aminoacridine Daunomycin 4-Nitroquinoline-N-oxide	E	Precipitation until end of experiment

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

Objective

The present study was conducted to investigate the test material for mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with β-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.

Results

Solvent and positive control treatments were included for all strains. The mean numbers of re-vertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following treatment of all bacteria tester strains with the test material in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test item was not mutagenic under the experimental conditions described.