1,1,3-trimethylindan-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:

11 - 26 Jul 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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:

- Only one plate was used per concentration - 2-Aminoanthracene was used as the sole indicator for the efficacy of the S9-mix

GLP compliance:

no

Remarks:

in-house safety study

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

EC Number:

838-724-1

Cas Number:

94568-76-0

Molecular formula:

C12H17N

IUPAC Name:

1,1,3-trimethyl-2,3-dihydro-1H-inden-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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Oriental Yeast Co., Ltd (Lot no. 12041901)

Test concentrations with justification for top dose:

First experiment: 15, 50, 150, 500, 1500, 5000 μg/plate with and without metabolic activation (recommended maximum test concentration for soluble non-cytotoxic substances according to OECD 471)
Additional tests; 15.6, 31.3, 62.5, 125, 250, 500 μg/plate with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: As the test substance does not dissolve in water at 50 mg/mL or higher, but did dissolve in DMSO at 50 mg/mL or higher and a 5% solution was stable for 24 hours at room temperature, DMSO was chosen as the solvent.

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, TA 98 0.1 µg/plate (-S9);
2) TA 1537 1.0 µg/plate (-S9);
3) TA 100 1.0 µg/plate TA 1535 TA 1537 2.0 µg/plate WP2uvrA10 µg/plate TA 98 0.5 µg/plate (+S9)
- a "negative control" not further specified was used

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): 1st experiment: one plate / dose; 2nd experiment: two plates / dose
- Number of independent experiments: 2 (in the second experiment, only the strain with a positive outcome was tested again)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added: 0.1 mL/plate; preincubation
The test substance was weighed, prepared and tested under yellow light.

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies per plate

Evaluation criteria:

Criteria for a positive result were dose dependency and a two-fold or greater increase in the number of revertant colonies in the group treated with the test substance compared to the number of revertant colonies in the negative control group. Otherwise the result was judged to be negative. If a positive result was found, comparative activity value was calculated.

Statistics:

Mean values were calculated.

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:

1st experiment: at 150 - 5000 μg/plate; 2nd experiment 250 and 500 µg/plate; precipitation in the 1st experiment at 5000 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:

1st experiment: at 150 - 5000 μg/plate; precipitation in the 1st experiment at 1500 µg/plate and higher

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1st experiment: at 500 - 5000 μg/plate; precipitation in the 1st experiment at 1500 µg/plate and higher (-S9) and at 5000 µg/plate (+S9)

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:

1st experiment: at 150 - 5000 µg/plate (-S9) and 500 - 5000 μg/plate (+S9); precipitation in the 1st experiment at 1500 µg/plate and higher (-S9) and at 5000 µg/plate (+S9)

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:

1st experiment: at 150 - 5000 μg/plate; precipitation in the 1st experiment at 1500 µg/plate and higher (-S9) and at 5000 µg/plate (+S9)

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1st experiment: at 500 - 5000 μg/plate; precipitation in the 1st experiment at 1500 µg/plate and higher (-S9) and at 5000 µg/plate (+S9)

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 (oil droplets) was observed in the first experiment at 1500 µg/plate and higher (-S9). No precipitation was observed in the second experiment.

STUDY RESULTS:
In the first experiment, there was a two-fold or greater increase in the number of revertant colonies observed for TA100 strain (+S9) compared to the negative control, and the increase was seen to be dose-dependent. The increase in the number of revertant colonies observed for TA100 strain (-S9), TA1535 strain (±S9), WP2 uvrA strain (±S9), TA98 strain (±S9) and TA1537 strain (±S9) were less than two-fold compared to the negative control. The positive controls induced a two-fold or greater increase in the number of revertant colonies compared to the negative control for each strain, showing the study was properly executed.
Bacterial growth inhibition by the test substance was found at 150, 500 or 5000 µg/plate with or without metabolic activation (depending on the strain).

A second experiment was carried out to replicate the positive result for TA100 strain (+S9). The result was again a two-fold or greater increase in the number of revertant colonies compared to the negative control, and the increase was found to be dose-dependent. The positive controls induced two-fold or greater increases in the number of revertant colonies compared to the negative control for each strain, showing the study was properly executed. Bacterial growth inhibition by the test substance was found at 250 and 500 µg/plate.
Thus, the results of the experiments showed that the positive result for TA100 strain (+S9) is replicable (please refer to the result tables under 'any other information on results incl. tables' for detailed information).

HISTORICAL CONTROL DATA
- Positive historical control data: not given in study report
- Negative (solvent/vehicle): not given in study report

Any other information on results incl. tables

Table 1: Results of the first experiment

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	86	10	31	14	4
		15	89	10	34	13	4
		50	89	10	26	14	4
		150	88*	9*	29	13*	3
		500	0*	0*	0*	0*	1*
		1500†	0*	0*	0*	0*	0*
		5000†	0*	0*	0*	0*	0*
S9 mix (+)		Negative control	130	9	34	21	17
		15	134	10	31	26	13
		50	223	7	36	27	11
		150	324*	6*	37	32	9
		500	0*	6*	20*	0*	3*
		1500†	0*	0*	0*	0*	0*
		5000†	0*	0*	0*	0*	0*
Positive controls	none	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	625	562	149	476	3220
	none	Name	2AA	2AA	2AA	2AA	2AA
		Dose (µg/plate)	1.0	2.0	10.0	0.5	2.0
		Colony count / plate	884	299	1098	319	135

* growth inhibition was noted
† precipitation was noted
Positive controls
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

 

 

Table 2: Results of the second experiment

Metabolic activation Y/N		Dose of test substance (μg/plate)	Number of reverse mutations (colony count/plate)
			Base pair substitution
			TA 100
S9 mix (–)		Negative control	97 103 (100)
S9 mix (+)		Negative control	128 115 (122)
		15.6	126 130 (128)
		31.3	200 193 (197)
		62.5	247 223 (235)
		125	294 302 (298)
		250	224* 225* (225)
		500	0* 0* (0)
Positive controls	none	Name	AF-2
		Dose (µg/plate)	0.01
		Colony count / plate	617 668 (643)
	none	Name	2AA
		Dose (µg/plate)	1.0
		Colony count / plate	896 931 (914)

Number in brackets is average value of colony count on each plate.
* growth inhibition was noted

Positive controls
AF-2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
2AA: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance 1,1,3-trimethylindan-4-amine has mutagenic potential (positive result).