1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

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:

22 Feb - 08 Mar 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

- As the outcome of this Ames Test is negative, a second confirmatory experiment is recommended (OECD 471). The preliminary test conducted here does not entirely fulfill this requirement, as it was performed according to the same protocol as the main experiment, and as other test concentrations were used. - Duplicates instead of triplicates were used in both experiments without provision of a scientific justification.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

- 2-Aminoanthracene was used as the sole indicator for the efficacy of the S9-mix

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

EC Number:

855-228-0

Cas Number:

4497-59-0

Molecular formula:

C14 H19 N O

IUPAC Name:

1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for the E.coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: tested for the presence / absence of amino acid requirements, sensitivity to UV radiation, membrane mutation rfa and the drug resistance factor R-factor

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tested for the presence / absence of amino acid requirements, sensitivity to UV radiation, membrane mutation rfa and the drug resistance factor R-factor

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Oriental Yeast Co., Ltd. (Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male SD rats treated with Phenobarbital and 5,6-Benzoflavone)
- method of preparation of S9 mix: S9 Mix was prepared at the time of use, and stored by ice cooling during use. Composition: S9: 0.1 mL; MgCl2: 8 µmol; KCl: 33 µmol; Glucose-6-phospate: 5 µmol; NADPH: 4 µmol; NADH: 4 µmol; Na-phosphate buffer (pH 7.4): 100 µmol
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix added to 0.1 mL of test substance solution; 0.1 mL S9 in 1 mL S9 mix preparation

Test concentrations with justification for top dose:

The doses were chosen based on the results of a dose-setting study carried out using each of the bacterial strains, with a maximum dose of 5000 µg/plate as specified in the guidelines, and serial dilution by a factor of 4 to give a total of 8 doses. Based on the effects in inhibiting growth of each bacterial strain, the doses below were set in the main study:

- TA100, TA1535 under conditions of S9(-): 4.9, 9.8, 20, 39, 78, 156, 313 µg/plate
- WP2 uvrA, TA98, TA1537 under conditions of S9(-): 9.8, 20, 39, 78, 156, 313 µg/plate
- TA100, TA1535, WP2 uvrA, TA98, TA1537 under conditions of S9(+): 9.8, 20, 39, 78, 156, 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The solubility of the test substance in water is < 50 mg/mL, but solubility in DMSO is ≥ 50 mg/mL and it was stable for 21 hours in a 5% solution at room temperature.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Remarks:

1) TA 100 0.01 µg/plate, WP2 uvrA 0.01 µg/plate, TA98 0.1 µg/plate (-S9)
2) TA 1537 1.0 µg/plate (-S9)
3) TA 100 1.0 µg/plate, TA 1535 2.0 µg/plate, WP2 uvrA 10.0 µg/plate, TA98 0.5 µg/plate, TA 1537 2.0 µg/plate (+S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 2 plates per dose for treated groups and pos. control; 3 plates for neg. control
- Number of independent experiments: 1 (Experiment was repeated due to contamination in +S9 conditions)

METHOD OF TREATMENT / EXPOSURE:
- Cell density at seeding: The bacterial concentrations were measured using as an indicator the absorbance of the culture solutions of each of the strains after 10-fold dilution with physiological saline using a spectrophotometer (measurement wavelength 660 nm, light path length 10 mm):
Strain TA100 TA1535 WP2 uvrA TA98 TA1537
Dose-setting study 3.2 4.4 6.5 3.4 2.7
Main study S9(-) 3.6 4.6 7.3 3.5 2.8
Main study (retest) S9(+) 3.7 4.8 6.7 3.5 2.6
[Figures indicating viable bacterial count (×10^9/mL)]
Test substance added: 0.1 mL/plate; preincubation method

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h at 37 °C

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Colony counting carried out using an automatic colony counter (CA-11 Automatic Colony Analyzer, System Science KK) with surface area correction and correction for counting errors by computer. When the number of colonies as the result of counting with the automatic colony counter is ≥ 1500, the precision of the automatic colony counter falls and so manual counting was carried out with a manual colony counter (manual colony counter No.3833, ANDERMAN & Co., Ltd.). Manual counting was also carried out when counting with the automatic counter was not possible because the colonies were very small due to strong growth inhibition. When the number of colonies was less than 300 with manual counting, all of these were counted and no correction was applied. When the number of colonies was ≥ 300, the plate was divided radially into 8 equal segments; 2 segments were counted and this number, corrected for surface area, was used.

Rationale for test conditions:

According to guideline ("Standards for Mutagenicity Testing Using Microorganisms"; Ordinance No. 77 (partially amended by MOL Ordinance No. 67 of 1997))

Evaluation criteria:

Based on the results of the dose-setting study, main study (S9(-)) and main study (retest) (S9(+)), an increase of ≥ 2 times in the revertant colony count in the groups treated with the test substance compared with the negative control group was assessed as a positive result when dose-dependence and reproducibility were observed. All other cases were assessed as negative. When a positive result was obtained, a specific activity value was calculated.

Statistics:

Statistical analysis was not used in evaluating the results of the study.
Mean values were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-/+): 313 µg/plate

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:

cytotoxicity

Remarks:

S9(-): 156, 313 µg/plate S9(+): 313 µg/plate

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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-/+): 313 µg/plate

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:

cytotoxicity

Remarks:

S9(-/+): 313 µg/plate

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:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-): 156, 313 µg/plate S9(+): 313 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance does not dissolve in water at 50 mg/mL or higher.
- Precipitation: Precipitation of the test substance did not occur.
- Other confounding effects: Growth of contaminating microorganisms was observed in S9 Mix in the test without bacteria in the main study, and therefore, colony counting under S9(+) conditions was not carried out, the plates were discarded, and the test was carried out again.

RANGE-FINDING/SCREENING STUDIES:
A dose-setting study was carried out using each of the bacterial strains, with maximum dose of 5000 µg/plate as specified in the guidelines, and serial dilutions by a factor of 4, to give a total of 8 doses (0.31; 1.2; 4.9; 20; 78; 313; 1250 and 5000 µg/plate). As a result, no increase of 2-fold or greater in revertant colonies was observed compared with the negative control, irrespective of the presence or absence of metabolic activation, for any of the strains. It should be noted that growth inhibition of each of the strains by this test substance was noted at the doses 313 - 5000 µg/plate for all strains with and without metabolic activation. Precipitation of the test substance did not occur.

STUDY RESULTS
No increase of 2-fold or greater in revertant colonies compared with the negative
control was noted, irrespective of the presence or absence of metabolic activation for any of the strains, and no dose-dependent increase was seen. It should be noted that, growth inhibition of each of the strains by this test substance was noted (see *-indicated results in table 2), irrespective of the presence or absence of metabolic activation; precipitation of the test substance was not observed.

HISTORICAL CONTROL DATA
- Positive historical control data: given in table 3; the positive controls induced revertant colonies more than twice the negative control for each strain, confirming validity of the response.
- Negative (solvent/vehicle) historical control data: In all of the tests, in the dose-setting study, the main study (S9(-)) and the main study (retest) (S9(+)), the negative control was within the permitted range of the background data for this institution (mean ± 2.5 SD).
The historical control data is provided under section "Any other information on results incl. tables, table 4"

Any other information on results incl. tables

Table 1: Test results of the dose-setting study

Study Period			February 22, 2013 to February 25, 2013
Metabolic activation Y/N		Test substance (µg/plate)	Revertant colonies/plate
			Base-pair substitution			Frame-shift
			TA100	TA1535	WP2 uvrA	TA98	TA1537
-S9Mix		Negative control	113 116 112 (114)	11 11 13 (12)	18 19 24 (20)	19 20 17 (19)	7 7 6 (7)
		0.31	101 118 (110)	12 14 (13)	23 17 (20)	14 14 (14)	3 4 (4)
		1.2	117 104 (111)	13 13 (13)	27 27 (27)	15 10 (13)	3 3 (3)
		4.9	126 108 (117)	8 10 (9)	23 19 (21)	11 14 (13)	4 4 (4)
		20	120 120 (120)	7 7 (7)	27 22 (25)	10 17 (14)	5 4 (5)
		78	117 112 (115)	6 6 (6)	18 23 (21)	17 23 (20)	5 4 (5)
		313	0* 0* (0)	0* 0* (0)	11* 14* (13)	13* 17* (15)	3* 4* (4)
		1250†	0* 0* (0)	0* 0* (0)	0* 0* (0)	0* 0* (0)	0* 0* (0)
		5000†	0* 0* (0)	0* 0* (0)	0* 0* (0)	0* 0* (0)	0* 0* (0)
+S9Mix		Negative control	136 131 135 (134)	7 7 8 (7)	27 25 24 (25)	22 20 27 (23)	14 15 17 (15)
		0.31	148 154 (151)	10 13 (12)	20 21 (21)	20 29 (25)	13 12 (13)
		1.2	149 132 (141)	6 6 (6)	24 21 (23)	30 33 (32)	12 13 (13)
		4.9	152 152 (152)	8 10 (9)	21 22 (22)	25 24 (25)	14 20 (17)
		20	127 150 (139)	12 8 (10)	27 24 (26)	26 23 (25)	15 14 (15)
		78	133 141  (137)	6 7 (7)	29 25 (27)	28 34  (31)	14 13 (14)
		313	69* 63* (66)	4* 5* (5)	22* 23* (23)	21* 18* (20)	13* 12* (13)
		1250	0* 0*  (0)	0* 0* (0)	0* 0* (0)	0* 0* (0)	0* 0*  (0)
		5000†	0* 0* (0)	0* 0*  (0)	0* 0* (0)	0* 0*  (0)	0* 0* (0)
Positive controls	No need for S9Mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
		Dose µg/plate	0.01	0.5	0.01	0.1	1.0
		Colony count/plate	483 487 (485)	518 428 (473)	100 111 (106)	391 394  ( 393)	1748 1880 (1814)
	Need for S9 mix	Name	2AA	2AA	2AA	2AA	2AA
		Dose µg/plate	1.0	2.0	10.0	0.5	2.0
		Colony count/plate	1017 1034  (1026)	318 280 ( 299)	612 688 ( 650)	351 323  ( 337)	133 154 ( 144)

Positive control substances: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN₃: sodium azide; ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride; 2AA: 2-aminoanthracene

*: concentrations inhibiting bacterial growth

†: Doses followed by † showed precipitation.

Numbers in brackets denote plate averages

 

 

 

Table 2: Test results of the main experiment (-S9)

Metabolic activation Y/N		Dose of test substance (μg/plate)	Revertant colonies/plate
			Base pair substitution			Frame shift
			TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
S9 mix (–)		Negative control	108 95 108 (104)	7 11 10 (9)	31 21 30 (27)	18 19 18 (18)	10 7 8 (8)
		4.9	116 100 (108)	7 7 (7)	-	-	-
		9.8	92 96 (94)	6 8 (7)	23 18 (21)	13 14 (14)	5 3 (4)
		20	98 112 (105)	10 8 (9)	21 24 (23)	17 21 (19)	3 4 (4)
		39	95 112 (104)	10 7 (9)	21 18 (20)	18 21 (20)	7 8 (8)
		78	87 93 (90)	11 12 (12)	23 25 (24)	19 16 (18)	5 6 (6)
		156	85* 100* (93)	10* 6* (8)	23 27 (25)	20 24 (22)	3 4 (4)
		313	55* 66* (61)	6* 4* (5)	15* 14* (15)	7* 8* (8)	3* 0* (2)
Positive controls	No need for S9 mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
		Dose (µg/plate)	0.01	0.5	0.01	0.1	1.0
		Colony count / plate	426 435 (431)	452 440 (446)	88 83 (86)	368 361 (365)	1928 2012 (1970)

Positive control substances: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN₃: sodium azide; ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride; 2AA: 2-aminoanthracene

*: are concentrations inhibiting bacterial growth

Numbers in brackets indicate plate averages

 

Table 3: Test results of the main study (retest; +S9)

Metabolic activation Y/N		Dose of test substance (μg/plate)	Revertant colonies/plate
			Base pair substitution			Frame shift
			TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
S9 mix (-)		Negative control	103 87 99 (96)	11 16 14 (14)	29 25 27 (27)	16 12 16 (15)	6 7 7 (7)
S9 mix (+)		Negative control	129 111 112 (117)	8 8 13 (10)	26 29 35 (30)	29 28 25 (27)	15 18 15 (16)
		9.8	128 114 (121)	13 11 (12)	28 34 (31)	20 24 (22)	16 15 (16)
		20	96 99 (98)	10 15 (13)	30 39 (35)	25 23 (24)	4 3 (4)
		39	115 136 (126)	8 10 (9)	34 29 (32)	23 27 (25)	15 13 (14)
		78	101 114 (108)	16 12 (14)	31 35 (33)	31 27 (29)	12 14 (13)
		156	97 93 (95)	13 9 (11)	35 42 (39)	28 25 (27)	14 12 (13)
		313	78* 0* (39)	0* 11* (6)	17* 18* (18)	14* 11* (13)	7* 8* (8)
Positive controls	No need for S9 mix	Name	AF-2	NaN3	AF-2	AF-2	ICR-191
		Dose (µg/plate)	0.01	0.5	0.01	0.1	1.0
		Colony count / plate	467 474 (471)	477 412 (445)	97 90 (94)	425 433 (429)	1315 1343 (1329)
	Need for S9 mix	Name	2AA	2AA	2AA	2AA	2AA
		Dose (µg/plate)	1.0	2.0	10.0	0.5	2.0
		Colony count / plate	852 955 (904)	271 246 (259)	767 800 (784)	314 296 (305)	117 96 (107)

Positive control substances: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN₃: sodium azide; ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride; 2AA: 2-aminoanthracene

* concentrations inhibiting bacterial growth

Numbers in brackets indicate plate averages

 

Table 4: Background data (Historical control data: September 7, 2012 to February 1, 2013)

TA100
Strain	Neg. ctrl. (S9-)	Neg. ctrl. (S9+)	Pos. ctrl (S9-) AF-2 (0.01 µg/plate)	Pos. ctrl (S9+) 2AA (1.0 µg/plate)
No. of data	64	64	64	64
Average	97	105	566	835
SD	10.1	12.8	60.8	80.3
TA1535
Strain	Neg. ctrl. (S9-)	Neg. ctrl. (S9+)	Pos. ctrl (S9-) NaN3 (0.5 µg/plate)	Pos. ctrl (S9+) 2AA (2.0 µg/plate)
No. of data	32	32	32	32
Average	12	11	491	257
SD	3.1	2.9	72.9	27.7
WP2 uvrA
Strain	Neg. ctrl. (S9-)	Neg. ctrl. (S9+)	Pos. ctrl (S9-) AF-2 (0.01 µg/plate)	Pos. ctrl (S9+) 2AA (10.0 µg/plate)
No. of data	32	32	32	32
Average	26	28	117	685
SD	5.3	6.0	15.4	101.7
TA98
Strain	Neg. ctrl. (S9-)	Neg. ctrl. (S9+)	Pos. ctrl (S9-) AF-2 (0.1 µg/plate)	Pos. ctrl (S9+) 2AA (0.5 µg/plate)
No. of data	103	103	103	103
Average	17	26	457	323
SD	2.9	4.3	40.7	39.3
TA1537
Strain	Neg. ctrl. (S9-)	Neg. ctrl. (S9+)	Pos. ctrl (S9-) ICR-191 (1.0 µg/plate)	Pos. ctrl (S9+) 2AA (2.0 µg/plate)
No. of data	33	33	33	33
Average	7	19	2588	118
SD	1.8	2.9	830.5	16.0

Positive control substances: AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; NaN₃: sodium azide; ICR-191: 6-chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride; 2AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

From the results in this study and under these test conditions, 1-(2,2,4-trimethyl-3,4-dihydroquinolin-l (2H)-yl)ethanone was assessed not to be mutagenic.