Triisononylamine

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:

From 19 December 2017 to 18 July 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 2016

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:

Triisononylamine

EC Number:

254-104-8

EC Name:

Triisononylamine

Cas Number:

38725-13-2

Molecular formula:

C27H57N

IUPAC Name:

tris(7-methyloctyl)amine

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch 99671
- Expiration date of the lot/batch: 9 October 2019
- Purity test date: 6 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: the test item was stored in the test facility in a closed vessel at room temperature (20±5°C)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: the test item was completely soluble in acetone, only
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No reactivity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: a stock solution containing 50 mL/L of the test item in acetone was prepared. Nominal concentrations were prepared
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: 50 mL/L
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
In solution with acetone

Method

Species / strain

Species / strain / cell type:

bacteria, other: S. Typhimurium TA97a, TA98, TA100, TA102 and TA1535

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other: See tables in "any other information on material and methods including tables" section

Metabolic activation:

with and without

Metabolic activation system:

S9 was obtained produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.

Test concentrations with justification for top dose:

On the day of the start of the experiment 1a, a stock solution containing 50 mL/L of the test item in acetone was prepared. The following nominal concentrations were prepared for the first experiment: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate.

The following nominal concentrations were prepared for the experiment 1b for the bacteria strains TA97a, TA98, TA100 and TA102: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate and 0.015 µL/plate.
The following nominal concentrations were prepared for the experiment 1b for the bacteria strain TA1535: 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate, 0.015 µL and 0.005 µL/plate.

The following nominal concentrations were prepared for the experiment 1b for the bacteria strains TA97a, TA98, TA100 and TA102: 5 µL/plate, 2.5 µL/plate, 1.25 µL/plate, 0.63 µL/plate, 0.31 µL/plate, 0.16 µL/plate, 0.08 µL/plate and 0.04 µL/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone.
The test item was completely soluble in acetone, only.
Acetone was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of sponta-neous revertants in the tested concentrations.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-nitro-1,2-phenylene Diamine ; 2-Amino-anthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): 10E9 bacteria/mL

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours after treatment

NUMBER OF REPLICATIONS: three replicates were used per condition

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were counted visually and the numbers were recorded.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: bacterial background lawn

Evaluation criteria:

A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. An increase factor of 3 should be taken into account for the bacteria strain TA1535. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: S. Typhimurium TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Experiment 1a:
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants (one exception) of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic muta-gens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations. In the highest concentration (5 µL/plate), no bacteria growth and no bacterial background lawn was observed. Towards the bacteria strain TA1535, signs of toxicity (decrease in the number of revertants) were observed in the next lower concentration (1.5 µL/plate), too. No increase of the number of revertant colonies in the treatments with and without meta-bolic activation could be observed. No concentration-related increase over the tested range was found.

Experiment 1b
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls (one exception in the negative control acetone) were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges. In this experiment, the test item showed no precipitates on the plates in all tested concentrations. In the highest concentration (5 µL/plate), no bacteria growth and no bacterial background lawn was observed. Towards the bacteria strain TA1535, signs of toxicity (decrease in the number of revertants) were observed in the next lower concentration (1.5 µL/plate), too. No increase of the number of revertant colonies in the treatments with and without meta-bolic activation could be observed. No concentration-related increase over the tested range was found.

Experiment 2:
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls (one exception in the negative control acetone) were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges. In this experiment, the test item showed no precipitates on the plates in all tested concentrations. Signs of toxicity were observed in the following concentrations with and without metabolic activation towards the resp. bacteria strains:
TA97a: no toxicity was observed
TA98: 5 µL/plate (decrease in the number of revertants)
TA100: 5 and 2.5 µL/plate (decrease in the number of revertants)
TA102: no toxicity was observed
TA1535: 1.5 µL/plate (decrease in the number of revertants)

The bacterial background lawn was visible in all concentrations.

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Historical data were presented in table in any other information on results including tables section.

Any other information on results incl. tables

Table2           Mean Revertants Experiment 1a

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	73	84	36	33	92	108	249	259	21	22
	sd	7.6	5.1	6.2	5.5	8.0	15.9	26.6	53.3	1.2	1.5
DMSO	Mean	74	110	38	36	89	91	239	249	26	18
	sd	5.2	21.1	4.4	11.9	9.2	1.2	28.9	46.0	1.7	0.6
Acetone	Mean	74	109	36	35	99	99	288	272	25	23
	sd	6.4	15.5	2.1	4.5	27.2	2.3	38.2	24.0	5.8	2.0
Positive Controls*	Mean	479	463	268	176	379	1001	657	1323	220	175
	sd	67.2	132.3	28.0	24.3	62.3	0.0	9.2	60.0	10.6	31.1
	f(I)	6.47	4.21	7.05	4.89	4.12	11.00	2.75	5.31	10.48	9.72
5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1.5 µL/plate	Mean	72	83	26	36	80	95	201	217	11	14
	sd	8.6	1.2	6.0	6.9	9.8	11.7	55.2	60.7	1.7	3.5
	f(I)	0.97	0.76	0.72	1.03	0.81	0.96	0.70	0.80	0.44	0.61
0.5 µL/plate	Mean	85	95	32	33	83	104	251	252	18	16
	sd	4.6	13.3	4.7	7.4	15.1	20.0	19.7	93.0	4.0	7.2
	f(I)	1.15	0.87	0.89	0.94	0.84	1.05	0.87	0.93	0.72	0.70
0.15 µL/plate	Mean	78	86	36	35	95	95	224	243	13	17
	sd	10.4	27.1	6.4	9.6	1.2	7.0	48.0	39.3	2.6	3.2
	f(I)	1.05	0.79	1.00	1.00	0.96	0.96	0.78	0.89	0.52	0.74
0.05 µL/plate	Mean	79	74	35	35	92	97	203	188	25	24
	sd	15.1	9.2	1.2	1.5	9.2	24.3	12.9	25.0	5.3	5.6
	f(I)	1.07	0.68	0.97	1.00	0.93	0.98	0.70	0.69	1.00	1.04

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

 

Table3Mean Revertants Experiment 1b

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	77	86	35	34	86	80	335	329	29	25
	sd	14.8	18.9	3.2	6.1	12.0	15.4	30.0	20.1	5.0	4.0
DMSO	Mean	79	71	34	38	75	87	328	283	21	19
	sd	15.6	9.5	2.6	6.5	14.2	20.7	36.0	35.9	2.6	5.9
Acetone	Mean	73	77	36	41	81	112	325	300	24	24
	sd	5.8	2.6	5.6	5.9	7.6	14.2	15.1	58.9	6.9	4.0
Positive Controls*	Mean	584	669	443	451	547	1001	1371	1533	343	243
	sd	65.5	6.1	72.6	68.0	196.3	0.0	59.0	53.3	44.1	11.5
	f(I)	7.39	9.42	13.03	11.87	6.36	11.51	4.18	5.42	11.83	12.79
5 µL/plate	Mean	72	89	8	11	20	36	41	43	n.d.	n.d.
	sd	12.0	11.8	1.0	1.0	6.1	20.8	0.6	8.7	n.d.	n.d.
	f(I)	0.99	1.16	0.22	0.27	0.25	0.32	0.13	0.14	n.d.	n.d.
1.5 µL/plate	Mean	84	101	34	34	79	106	320	293	7	4
	sd	4.0	14.5	7.6	6.4	3.6	18.6	52.5	134.5	0.6	2.3
	f(I)	1.15	1.31	0.94	0.83	0.98	0.95	0.98	0.98	0.29	0.17
0.5 µL/plate	Mean	88	119	34	35	82	88	231	340	30	30
	sd	8.0	4.2	4.4	4.5	10.1	16.5	56.6	66.1	9.2	6.7
	f(I)	1.21	1.55	0.94	0.85	1.01	0.79	0.71	1.13	1.25	1.25
0.15 µL/plate	Mean	94	97	33	39	82	105	311	328	29	31
	sd	1.7	6.4	7.0	10.4	12.8	6.4	112.0	0.0	9.1	7.0
	f(I)	1.29	1.26	0.92	0.95	1.01	0.94	0.96	1.09	1.21	1.29
0.05 µL/plate	Mean	70	89	43	38	111	112	293	395	35	28
	sd	12.1	18.6	1.5	6.4	13.0	14.2	48.9	51.4	4.6	7.5
	f(I)	0.96	1.16	1.19	0.93	1.37	1.00	0.90	1.32	1.46	1.17
0.015 µL/plate	Mean	78	76	36	36	97	100	384	403	26	31
	sd	8.7	8.7	1.7	5.0	15.1	18.3	52.5	41.1	3.0	6.1
	f(I)	1.07	0.99	1.00	0.88	1.20	0.89	1.18	1.34	1.08	1.29
0.005 µL/plate	Mean	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	32	34
	sd	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	6.7	6.4
	f(I)	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	n.d.	1.33	1.42

1001 colonies per plate means the bacteria growth was too strong for counting.

n.d. = not determined, due to the toxicity effect in experiment 1a

 f(I) = increase factor

 

Table4          Mean Revertants Experiment 2

Strain		TA97a		TA98		TA100		TA102
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	80	75	34	45	101	117	317	325
	sd	14.0	8.3	4.0	2.1	14.2	2.3	22.7	18.0
DMSO	Mean	71	81	41	42	87	92	304	300
	sd	9.0	1.2	7.5	3.8	5.9	10.4	38.2	41.6
Acetone	Mean	86	79	39	42	84	120	331	291
	sd	13.5	12.5	5.3	3.6	3.5	5.3	10.1	36.3
Positive Controls*	Mean	268	368	307	328	372	1001	663	1267
	sd	50.1	26.2	59.9	52.9	32.7	0.0	52.8	125.6
	f(I)	3.77	4.54	7.49	7.81	3.68	10.88	2.18	4.22
5 µL/plate	Mean	72	93	15	12	12	26	296	343
	sd	19.1	25.4	1.2	5.2	1.0	9.9	55.0	20.1
	f(I)	0.84	1.18	0.38	0.29	0.14	0.22	0.89	1.18
2.5 µL/plate	Mean	78	89	37	24	38	45	267	256
	sd	12.5	5.3	7.5	5.6	13.5	13.1	82.6	24.3
	f(I)	0.91	1.13	0.95	0.57	0.45	0.38	0.81	0.88
1.25 µL/plate	Mean	77	74	37	29	74	76	333	369
	sd	4.7	7.5	5.2	10.0	10.5	6.4	24.1	100.4
	f(I)	0.90	0.94	0.95	0.69	0.88	0.63	1.01	1.27
0.63 µL/plate	Mean	69	77	36	34	75	104	316	329
	sd	14.2	3.1	4.2	3.0	4.5	8.5	50.0	56.6
	f(I)	0.80	0.97	0.92	0.81	0.89	0.87	0.95	1.13
0.31 µL/plate	Mean	100	77	34	38	86	96	320	376
	sd	10.1	9.6	8.4	3.5	8.7	15.0	48.5	17.4
	f(I)	1.16	0.97	0.87	0.90	1.02	0.80	0.97	1.29
0.16 µL/plate	Mean	82	72	35	33	111	110	352	388
	sd	1.5	3.2	14.0	3.8	12.7	11.7	24.3	82.7
	f(I)	0.95	0.91	0.90	0.79	1.32	0.92	1.06	1.33
0.08 µL/plate	Mean	86	91	37	34	108	105	397	421
	sd	18.0	4.0	2.5	1.7	8.9	18.0	73.4	26.6
	f(I)	1.00	1.15	0.95	0.81	1.29	0.88	1.20	1.45
0.04 µL/plate	Mean	80	81	42	36	93	89	297	389
	sd	2.0	1.0	12.5	2.6	16.8	8.7	75.6	68.6
	f(I)	0.93	1.03	1.08	0.86	1.11	0.74	0.90	1.34

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

 

Strain		TA1535
Induction		-S9	+S9
Demin. water	Mean	14	12
	sd	5.2	1.7
DMSO	Mean	11	16
	sd	5.1	4.2
Acetone	Mean	16	15
	sd	2.6	5.1
Positive Controls*	Mean	333	197
	sd	56.8	76.4
	f(I)	23.79	12.31
1.5 µL/plate	Mean	5	3
	sd	0.6	0.6
	f(I)	0.31	0.20
0.75 µL/plate	Mean	13	14
	sd	3.6	2.5
	f(I)	0.81	0.93
0.38 µL/plate	Mean	14	12
	sd	1.0	2.5
	f(I)	0.88	0.80
0.19 µL/plate	Mean	12	15
	sd	1.5	3.1
	f(I)	0.75	1.00
0.09 µL/plate	Mean	13	8
	sd	2.9	1.2
	f(I)	0.81	0.53
0.05 µL/plate	Mean	13	11
	sd	4.2	2.6
	f(I)	0.81	0.73
0.02 µL/plate	Mean	13	14
	sd	2.9	3.2
	f(I)	0.81	0.93
0.01 µL/plate	Mean	17	13
	sd	4.6	1.5
	f(I)	1.06	0.87

f(I) = increase factor

Table5           Historical Data of Spontaneous Revertants

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
Demin. water	Mean	89	94	20	22	92	96	279	297	18	18
	Min	60	63	6	8	51	64	85	67	6	7
	Max	144	138	52	51	141	141	425	511	31	33
	SD	18	16	11	10	15	14	58	69	6	6
	Exp 1a	73	84	36	33	92	108	249	259	21	22
	Exp 1b	77	86	35	34	86	80	335	329	29	25
	Exp 2	80	75	34	45	101	117	317	325	14	12
DMSO	Mean	88	97	20	21	89	92	278	294	18	17
	Min	58	67	7	8	44	62	79	80	8	6
	Max	135	144	47	50	136	199	393	459	33	32
	SD	17	17	11	10	16	17	56	61	6	6
	Exp 1a	74	110	38	36	89	91	239	249	26	18
	Exp 1b	79	71	34	38	75	87	328	283	21	19
	Exp 2	71	81	41	42	87	92	304	300	11	16
Acetone	Mean	66	107	16	19	68	76	335	338	16	16
	Min	34	61	6	11	52	54	227	223	10	10
	Max	88	139	52	51	109	106	525	485	24	25
	SD	17	25	11	10	14	14	83	83	4	4
	Exp 1a	74	109	36	35	99	99	288	272	25	23
	Exp 1b	73	77	36	41	81	112	325	300	24	24
	Exp 2	86	79	39	42	84	120	331	291	16	15
Positive Controls*	Mean	537	520	407	113	490	764	1110	1213	265	132
	Min	264	237	100	39	220	273	491	408	55	45
	Max	1165	1181	1001	487	984	1912	2331	6083	515	712
	SD	173	160	163	90	153	288	423	585	88	82
	Exp 1a	479	463	268	176	379	1001	657	1323	220	175
	Exp 1b	584	669	443	451	547	1001	1371	1533	343	243
	Exp 2	268	368	307	328	372	1001	663	1267	333	197

* Different positive controls were used

1001 colonies per plate means the bacteria growth was too strong for counting.

 

Applicant's summary and conclusion

Conclusions:

Under the experimental condition of the study, the results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Hence, the test item Triisononylamine was not considered as mutagenic for bacteria strain according to CLP criteria.

Executive summary:

In this GLP compliant in vitro test, the test item Triisononylamine was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) according to OECD TG 471 method.

The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

Experiment 1a:

In this experiment, the test item (dissolved in acetone) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. In the highest concentration (5 µL/plate), no bacteria growth and no bacterial background lawn was observed. Towards the bacteria strain TA1535, signs of toxicity (decrease in the number of revertants) were observed in the next lower concentration (1.5 µL/plate), too.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 1b:

In this experiment (repetititon of experiment 1a with lower concentrations), the test item was tested up to concentrations of 5  µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100 and TA102 and up to concentrations of 1.5  µL/plate in the absence and presence of S9-mix in the strain TA1535 using the plate incorporation method, too. The test item showed no precipitates on the plates at any of the concentrations. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2:

Based on the toxicity results of experiment 1a and 1b, the test item was tested up to con-centrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100 and TA102 and up to concentrations of 1.5  µL/plate in the absence and presence of S9-mix in the strain TA1535 using the pre-incubation method.

Signs of toxicity were observed in the following concentrations with and without metabolic activation towards the resp. bacteria strains:

TA97a: no toxicity was observed

TA98: 5 µL/plate (decrease in the number of revertants)

TA100: 5 and 2.5 µL/plate (decrease in the number of revertants)

TA102: no toxicity was observed

TA1535: 1.5 µL/plate (decrease in the number of revertants)

The results of this experiments showed that the test item caused no increase in the num-ber of revertants in all bacteria strains compared to the solvent control, in both the ab-sence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that Triisononylamine is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study. Hence, the test item Triisononylamine was not considered as mutagenic for bacteria strain according to CLP criteria.