(3R)-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

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:

05 - 14 Mar 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

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 (incl. QA statement)

Remarks:

Certifying authority: Ministry of Health, Labour, and Welfare, Japan.

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(3R-)1,1,3-trimethylindan-4-amine

EC Number:

836-285-0

Cas Number:

125349-37-3

Molecular formula:

C12H17N

IUPAC Name:

(3R-)1,1,3-trimethylindan-4-amine

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.
- method of preparation of S9 mix: The S9 (Lot No. 12113009, manufactured on 30 Nov 2012) was prepared from the livers of 7 week-old male Sprague Dawley rats weighing 206.9 ± 11.3 g (Mean ± S.D.). These rats had been treated with phenobarbital (PB) and 5,6-benzoflavone (BF). Prior to the S9 preparation, PB was intraperitoneally injected 4 times (Day 1: 30 mg/kg bw and Days 2 - 4: 60 mg/kg bw) and BF was intraperitoneally injected once on Day 3 (80 mg/kg bw). The S9 was stored at -80°C from purchase until use. The S9 mix was prepared by mixing thawed S9 with sterilized co-factors mildly, and maintained on ice-bath until use. Composition (amount in 1 mL of S9 mix): S9: 0.1 mL; MgCl2: 8 µmol; KCl: 33 µmol; Glucose-6-phosphate: 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 test substance solution; the S9 mix contained 10 v/v% of S9 (0.1 mL S9 in 1 mL of S9 mix)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The sterile conditions of S9 mix and the test substance solution were checked by conduction of a sterility test.

Test concentrations with justification for top dose:

The concentrations were based on the results of a dose-setting test and set as follows:
For TA 100 strain with metabolic activation: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 µg/plate
For TA1535, TA98, TA1537, WP2uvrA with metabolic activation and for TA100, TA1535, TA98, TA1537, WP2 uvrA without metabolic activation: 313, 156, 78.1, 39.1, 19.5, 9.77 µg/plate

In the pre-test, cytotoxicity above dose levels of 313 µg/plate and an increased number of revertant colonies were noted for TA 100 with metabolic activation. Hence, dose levels were set up to incl. 313 µg/plate as the top dose, and not up to the maximum dosage of 5000 µg/plate as outlined in OECD 471.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO) (also for all positive control substances)
- Justification for choice of solvent/vehicle: The test substance was not soluble in water at 50 mg/mL, but it was soluble in DMSO at 50 mg/mL, and it was stable in DMSO at 5% for 6 h 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:

9-aminoacridine

sodium azide

other:

Remarks:

1) TA 100 0.01 µg/plate, WP2 uvrA 0.01 µg/plate, TA 98 0.1 µg/plate (-S9)
2) TA 100 1.0 µg/plate, TA 1535 2.0 µg/plate, WP2 uvrA 10 µg/plate, TA 98 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): Duplicate plates were used for each concentration.
- Number of independent experiments: 1

METHOD OF TREATMENT / EXPOSURE:
- Cell density at seeding: The number of viable cells for each culture was calculated from the turbidity measured by a spectrophotometer and are as follows:
Strain TA100 TA1535 WP2 uvrA TA98 TA1537
Dose-setting study 3.17 2.97 5.08 2.66 1.96
Main study 3.29 3.05 4.60 2.67 1.97
[Figures indicating viable bacterial count (×10^9/mL)]
- Test substance added: 0.1 mL/plate; pre-incubation method

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate (cytotoxicity to bacteria by the test substance was examined under a stereomicroscope (x40))

METHODS FOR MEASUREMENT OF GENOTOXICITY
Colony counting was performed using a colony analyzer CA-11S (System Science Co., Ltd.) and the counted values were adjusted to correct both the area and counting loss.

DETERMINATION OF PRECIPITATION
Precipitation of the test substance was observed by unaided eyes.

Rationale for test conditions:

Ministry of Labor, Japan: "Mutagenicity tests in Industrial Safety and Health Law, Japan - Testing guidelines and GLP"; Japan Industrial Safety and Health Association (1991)

Evaluation criteria:

When the test substance induces a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the negative control and the increase is reproducible, the test substance is judged positive int his test system.

Statistics:

Mean values were calculated.
Statistical analysis was not performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 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:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 156 and 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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 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 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 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 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

S9(-): at 156 and 313 µg/plate S9(+) at 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 was not observed at any dose levels with or without metabolic activation in the dose-finding test and the main test.
- Sterility test: To test for sterile conditions, the S9 mix and the test substance solution were spread on a minimal glucose agar plate using a top agar solution, and after incubation for 48 h at 37 °C, the plates were checked for presence or absence of bacterial contamination by unaided eyes. No contamination was observed.

RANGE-FINDING/SCREENING STUDIES:
In order to set the dose levels for the main test, a dose-finding test was performed with the highest dose level of 5000 µg/plate, and at eight dose levels in total (0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate). The number of revertant colonies was counted. Cytotoxicity to bacteria by the test substance and the precipitation of the test substance were examined. The test substance induced increase in the number of revertant colonies to at least twice as many as that of the negative control for TA100 strain with metabolic activation, and the dose-dependency was observed. The test substance did not induce any increases in the number of revertant colonies to at least twice as many as that of the negative control for TA1535, TA98, TA1537, WP2uvrA with metabolic activation or for TA100, TA1535, TA98, TA1537, WP2uvrA without metabolic activation. Cytotoxicity to bacteria by the test substance was observed at and above dose levels of 313 µg/plate with and without metabolic activation. Precipitation of the test substance was not observed any dose levels with or without metabolic activation.

STUDY RESULTS:
Relative mutagenic activity was calculated for doses that caused ≥ 2-fold increases in the number of revertant colonies relative of the negative control, and the following formula was used:
Relative mutagenic activity (revertants/mg) = [[(Number of colonies per plate at the pertinent dose)-(Number of colonies in negative control test)] / (Dose used (µg))] * 1000
For the positive result of TA100 under S9(+) conditions, the highest relative mutagenic activity was calculated to be 3.0*10^3 revertants/mg (at 39.1 µg/plate for TA100 with metabolic activation in the main test).

Throughout the tests, the test substance induced the dose-dependent increase in the number of revertant colonies to at least twice as many as that of the negative control for TA100 strain with metabolic activation, and the dose-dependent increase was reproducible.
For TA1535, TA98, TA1537, WP2 uvrA with metabolic activation or for TA100, TA1535, TA98, TA1537, WP2 uvrA without metabolic activation, test substance did not induce any dose-dependent increases in the number of revertant colonies to at least twice as many as that of the negative control. Therefore, the test substance was concluded to be mutagenic under the conditions of this test. The highest relative mutagenic activity is 3.0*10^3 revertants/mg (at 39.1 µg/plate for TA100 with metabolic activation in the main test).
In absence of metabolic activation, cytotoxicity was noted at 156 µg/plate for TA 100 and TA 1535 and at 313 µg/plate in all tester strains. In presence of metabolic activation, cytotoxicity was noted at 313 µg/plate in all tester strains. Precipitation of the test substance was not observed at any dose level with or without metabolic activation.

HISTORICAL CONTROL DATA
Positive and negative control data were given. In the dose-finding test and the main test, the numbers of revertant colonies of negative and positive controls for any of the bacterial strains were within the ranges of the reference values (the mean ± 3SD) calculated on the historical data in the testing facility. The historical control data is provided in table 2 under section "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Results of the main study

Metabolic activation Y/N		Dose of test substance (μg/plate)	Number of reverse mutations (colony count/plate)
			Base pair substitution			Frame shift
			TA 100	TA 1535	WP2 uvrA	TA 98	TA 1537
S9 mix (–)		Negative control	95 89 (92)	13 9 (11)	19 24 (22)	11 10 (11)	5 5 (5)
		9.77	96 91 (94)	8 6 (7)	19 15 (17)	15 15 (15)	5 5 (5)
		19.5	92 97 (95)	7 9 (8)	16 16 (16)	8 11 (10)	3 5 (4)
		39.1	107 84 (96)	8 8 (8)	18 20 (19)	13 9 (11)	8 5 (7)
		78.1	95 101 (98)	14 4 (9)	24 28 (26)	14 9 (12)	5 4 (5)
		156	68* 80* (74)	5* 10* (8)	18 19 (19)	13 10 (12)	4 6 (5)
		313	75* 66* (71)	4* 7* (6)	23* 16* (20)	6* 9* (8)	6* 4* (5)
S9 mix (+)		Negative control	98 116 (107)	6 10 (8)	24 36 (30)	20 27 (24)	10 10 (10)
		4.88	107 124 (116)
		9.77	141 134 (138)	7 10 (9)	20 28 (24)	16 15 (16)	8 9 (9)
		19.5	153 177 (165)	10 9 (10)	28 25 (27)	25 25 (25)	4 9 (7)
		39.1	237 208 (254)	10 5 (8)	27 26 (27)	32 25 (29)	11 4 (8)
		78.1	268 240 (254)	10 13 (12)	32 20 (26)	23 28 (26)	11 10 (11)
		156	271 269 (270)	6 10 (8)	18 23 (21)	20 23 (22)	10 11 (11)
		313	212* 240* (226)	5* 4* (5)	20* 22* (21)	14* 23* (19)	10* 10* (10)
Positive controls	S9 mix (-)	Name	AF-2	NaN3	AF-2	AF-2	9-AA
		Dose (µg/plate)	0.01	0.5	0.01	0.1	80
		Colony count / plate	561 546 (554)	384 416 (400)	146 151 (149)	422 435 (429)	240 279 (260)
	S9 mix (+)	Name	2AA	2AA	2AA	2AA	2AA
		Dose (µg/plate)	1.0	2.0	10.0	0.5	2.0
		Colony count / plate	1125 1093 (1109)	332 362 (347)	623 707 (665)	439 374 (407)	189 208 (199)

1)AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
2)NaN3: sodium azide
3)9-AA: 9-Aminoacridine
4)2-AA: 2-Aminoanthracene

* cytotoxicity was noted
The average number of revertants in each dose are shown in parentheses.

 

Table 2: Historical data and reference values of negative and positive controls

		Negative control				Positive control
Bacterial strain	S9 (+) or S9 (-)	Historical data			Reference Value*	Substance and dose	Historical data			Reference Value*
		Mean	SD^1)	No. of data			Mean	SD	No. of data
TA98	S9 (-)	15	3	308	5 - 25	AF-2^2) 0.1 µg/plate	529	59	304	353 - 706
	S9 (+)	24	5	298	9 - 39	2-AA^3) 0.5 µg/plate	319	68	294	114 - 524
TA100	S9 (-)	91	13	306	53 - 129	AF-2 0.01 µg/plate	665	75	302	439 - 892
	S9 (+)	107	14	296	64 - 150	2-AA 1.0 µg/plate	968	153	292	509 - 1428
TA1535	S9 (-)	9	2	286	1 - 16	NaN3^4) 0.5 µg/plate	363	48	282	220 - 506
	S9 (+)	9	3	278	1 - 17	2-AA 2.0 µg/plate	289	54	274	126 - 453
TA1537	S9 (-)	6	2	286	1 - 13	2-AA^5) 80 µg/plate	205	61	282	26 - 389
	S9 (+)	11	3	284	2 - 20	2-AA 2.0 µg/plate	145	30	280	55 - 235
WP2 uvrA	S9 (-)	24	8	266	1 - 48	AF-2 0.01 µg/plate	144	22	266	96 - 211
	S9 (+)	28	9	262	2 - 54	2-AA 10 µg/plate	775	134	262	371 - 1178

* The ranges of the mean ± 3SD of tests performed by pre-incubation method from July 2012 to December 2012 in the testing facility. When the value of mean - 3SD in the negative control was less than 1, the lower limit of the reference value was set as 1. When the value of mean - 3SD in the positive control was less than twice as many as the value of mean + 3SD in the negative control at the same condition, the lower limit of the reference value in the positive control was set twice as many as the value of mean + 3SD in the negative control at the same condition.

1) SD: Standard deviation

2) AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

3) 2AA: 2-aminoanthracene

4) NaN₃: sodium azide

5) 9AA: 9-Aminoacridine

Applicant's summary and conclusion

Conclusions:

The test substance (3R-)2,3-dihydro-1,1,3-trimethyl-1H-inden-4amine was concluded to be mutagenic under the conditions of this test. The highest relative mutagenic activity is 3.0*10^3 revertants/mg (at 39.1 µg/plate for TA100 with metabolic activation in the main test.