1,2-Propanediamine, N1-(2-aminoethyl)-3-(4-ethoxyphenyl)-, hydrochloride (1:2), (2S)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / Ames test): negative with and without metabolic activation Gürtler & Görke 1997

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from November to December 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Deviations:

yes

Remarks:

- TA 102 or E.coli WP2 strains are not included; only one experiment (direct plate incorporation procedure) was performed

Principles of method if other than guideline:

The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male Wistar rat liver S9 mix

Test concentrations with justification for top dose:

100, 250, 500, 1000, 2500, 5000 µg/plate

Vehicle / solvent:

phosphate buffer ((pH 7.4, 0.1 M)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

cyclophosphamide

other: anthracen-2-amine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration triplicate
- Number of independent experiments; 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E-06 dilution
- Test substance added in medium; in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically.

Evaluation criteria:

The plates were scored for the number of mutant colonies with an automated colony counter (ArtekM 982B, Artek Systems Corporation, Farrningdale, NY, USA). ln exceptional cases, where reliable automatic counting is not possible, e.g. due to distinct precipitates of the test compound, the colonies are scored manually. The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if the number of revertants of the compound groups compared to the number of revertants of the negative control group was reproducibly higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect.

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Water solubility: no limited water solubility reported
- Precipitation and time of the determination: no precipitates

RANGE-FINDING/SCREENING STUDIES (if applicable): not performed, tested up to the highest recommended concentration

STUDY RESULTS
- Concurrent vehicle negative and positive control data: yes


Ames test:
- Signs of toxicity: No
- Individual plate counts: please refer to 'Any other information on results incl. tables'
- Mean number of revertant colonies per plate and standard deviation: please refer to 'Any other information on results incl. tables'

None of the five tester strains showed increased reversion to prototrophy in the assays at 6 concentrations tested between 0.1 and 5.0 mg/plate, either in the absence or presence of S9 mix. Generally, growth inhibition of the background lawn was not observed. There were no precipitates in the agar. Negative controls and positive controls with known mutagens produced the expected numbers of revertant colonies.

TA 1535

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
Phosphate buffer	50 µL	24	26	3	13	16	3	1.0	1.0
		25			19
		30			17
DMSO	50 µL	42	27	13	16	16	4	1.0	1.0
		18			19
		22			12
Test item	0.10 mg	24	28	4	12	12	1	1.1	0.7
		32			12
		29			11
Test item	0.25 mg	28	26	2	21	19	3	1.0	1.1
		27			19
		24			18
Test item	0.5 mg	28	25	6	17	15	4	1.0	0.9
		29			18
		19			11
Test item	1.0 mg	27	26	4	10	13	3	1.0	0.8
		30			16
		22			12
Test item	2.5 mg	25	28	3	15	16	2	1.1	1.0
		31			15
		29			19
Test item	5.0 mg	30	27	4	14	12	3	1.0	0.7
		23			12
		29			9
Anthracen-2-amine	2 µg	33	31	3	105	118	11	1.2	7.2
		32			126
		28			122
Cyclophosphamide	400.0 µg	49	53	13	463	451	13	2.0	27.6
		42			454
		67			437
Sodium azide	5.0 µg	627	622	6	201	177	21	23.6	10.8
		624			165
		615			164

 

TA100

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
Phosphate buffer	50 µL	110	114	3	134	121	12	1.0	1.0
		116			119
		115			111
DMSO	50 µL	143	120	20	92	100	8	1.1	0.8
		109			99
		109			108
Test item	0.1 mg	129	122	12	88	105	18	1.1	0.9
		109			105
		129			123
Test item	0.25mg	134	118	15	110	101	9	1.0	0.8
		105			93
		114			101
Test item	0.5mg	123	122	17	96	102	6	1.1	0.8
		105			105
		138			106
Test item	1.0 mg	117	117	6	107	101	5	1.0	0.8
		111			98
		123			98
Test item	2.5 mg	122	108	18	116	115	3	0.9	1.0
		87			112
		114			118
Test item	5.0 mg	48 B	44	3	104	102	2	0.4	0.8
		43			100
		42			101
Anthracen-2-amine	2.0 µg	110	111	4	662	747	82	1.0	6.2
		107			755
		115			825
Benzo[a]pyrene	2.5 µg	107	104	11	573	571	39	0.9	4.7
		92			531
		113			608
Sodium azide	5.0 µg	669	694	43	206	215	8	6.1	1.8
		669			221
		743			217

 

TA1537

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
Phosphate buffer	50 µL	14	13	3	14	17	4	1.0	1.0
		15			15
		9			22
DMSO	50 µL	14	11	3	13	16	5	0.9	0.9
		10			22
		9			13
Test item	0.1 mg	14	10	4	10	17	7	0.8	1.0
		8			16
		7			24
Test item	0.25 mg	10	8	2	18	15	3	0.7	0.9
		7			15
		8			13
Test item	0.5 mg	11	12	4	21	19	2	0.9	1.1
		9			17
		16			18
Test item	1.0 mg	13	13	4	18	18	1	1.1	1.0
		17			17
		10			18
Test item	2.5 mg	16	17	1	18	18	2	1.3	1.1
		17			17
		17			20
Test item	5.0 mg	14	13	2	13	13	0	1.0	0.8
		10			13
		14			13
Anthracen-2-amine	2.0 µg	14	12	3	85	76	13	0.9	4.5
		14			61
		8			83
9-Aminoacidine	100.0 µg	1018	987	44	784	872	76	77.9	51.3
		1005			917
		937			914

 

TA1538

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
Phosphate buffer	50 µL	12	9	3	22	22	7	1.0	1.0
		6			15
		8			28
DMSO	50 µL	16	13	3	23	24	1	1.5	1.1
		11			23
		12			25
Test item	0.1 mg	10	9	1	30	29	1	1.1	1.4
		10			30
		8			28
Test item	0.25 mg	12	12	2	24	25	4	1.4	1.1
		14			21
		10			29
Test item	0. 5 mg	12	12	2	27	25	7	1.4	1.1
		10			17
		14			30
Test item	1.0 mg	4	7	3	20	27	6	0.8	1.2
		10			31
		7			29
Test item	2.5 mg	16	12	4	36	35	2	1.4	1.6
		8			33
		12			35
Test item	5.0 mg	10	12	2	33	33	7	1.4	1.5
		12			40
		14			27
Anthracen-2-amine	2.0 µg	8	8	2	583	591	13	1.0	27.3
		10			583
		7			606
2-Nitrofluorene	10.0 µg	1128	1124	66	385	389	5	129.7	18.0
		1188			387
		1057			395

 

TA98

Substance and dose/plate		Revertants per plate						Quotient
		-S9	M	±SD	+S9	M	±SD	-S9	+S9
Phosphate buffer	50 µL	20	21	6	51	49	3	1.0	1.0
		27			49
		15			46
DMSO	50 µL	25	20	4	41	50	8	1.0	1.0
		17			55
		19			53
Test item	0.1 mg	14	19	6	51	48	6	0.9	1.0
		25			51
		17			41
Test item	0.25 mg	19	17	3	41	53	10	0.8	1.1
		19			56
		13			61
Test item	0.5 mg	16	21	7	45	51	7	1.0	1.0
		29			49
		17			59
Test item	1.0 mg	30	30	3	46	49	4	1.5	1.0
		33			49
		27			53
Test item	2.5 mg	19	23	10	67	60	8	1.1	1.2
		16			52
		34			62
Test item	5.0 mg	14	16	4	41	47	8	0.8	1.0
		14			43
		21			56
Anthracen-2-amine	2.0 µg	19	20	2	630	615	19	1.0	12.6
		18			594
		22			621
Benzo[a]pyrene	2.5 µg	21	17	5	317	323	7	0.8	6.6
		18			330
		12			322
2-Nitrofluorene	10.0 µg	1042	1075	33	342	401	57	52.0	8.2
		1076			455
		1107			407

 

M=mean

B= Background lawn reduced

± SD=Standard Deviation

+S9= With S9 mix

-S9=Without S9 mix

Quotient= Mean revertants (test substance)/Mean revertants (Solvent)

 

Conclusions:

The mutagenic potential of the test substance was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the presence and absence of S9 mix according to OECD TG 471. Evidence of mutagenic activity was not seen up to the maximum recommended dose level of 5000 µg/plate. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level in the presence and absence of metabolic activation. Therefore, the test substance was considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May, 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Triamine Dihydrochloride in phosphate buffer at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.

The test item was tested limit concentration 5000 µg/plate All of the five tester strains showed no increased reversion to prototrophy at all concentrations tested, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was observed only at the highest concentration tested (5000 µg/plate) in the TA100 strain without metabolic activation. There were no precipitates in the agar found at any concentration in all strains used in the tests without and with S9 mix.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Evaluation of the data does indicate that the test substance is not a mutagen in the Ames-Test. In the five tester strains (all strains of Salmonella typhimurium) no increased reversion to prototrophy with the test substance at the doses tested in the absence and presence of S9 mix were observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 26 May, 1983), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to Triamine Dihydrochloride in phosphate buffer at concentrations of 100, 250, 500, 1000, 2500, and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.

The test item was tested limit concentration 5000 µg/plate. All of the five tester strains showed no increased reversion to prototrophy at all concentrations tested, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was observed only at the highest concentration tested (5000 µg/plate) in the TA100 strain without metabolic activation. There were no precipitates in the agar found at any concentration in all strains used in the tests without and with S9 mix.

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Evaluation of the data does indicate that the test substance is not a mutagen in the Ames-Test. In the five tester strains (all strains of Salmonella typhimurium) no increased reversion to prototrophy with the test substance at the doses tested in the absence and presence of S9 mix were observed.

 

In a QSAR prediction using DEREK Nexus v6.1 the potential of Triamine Dihydrochloride to induce mutagenicity was assessed. Derek Nexus makes qualitative predictions for and against toxicity through reasoning. For the endpoint of mutagenicity, predictions for toxicity decrease in confidence in the following order: certain>probable>plausible>equivocal. Predictions against toxicity increase in confidence in the following order: inactive (with unclassified and/or misclassified features)<inactive<improbable. Likelihood levels have beenshown to correlate with predictivity [Judson et al, 2013]. Multiple data sources (e.g. toxicity data from multiple assays and mechanistic evidence) are synthesised into the structure-activity relationships that underpins Derek Nexus predictions. An appreciation of the assay units applied by alert writers when building the alert training set. However, predictions are not quantitative and, as a result, do not include units.

The query structure does not match any structural alerts or examples for (bacterial in vitro) mutagenicity in Derek. Furthermore, the query structure does  not contain an unclassified feature and is consequently not predicted to be indeterminate in the bacterial in vitro (Ames) mutagenicity test. However, experimental data are available clearly reporting a negative result.

Based on these results Triamine Dihydrochloride is considered non-mutagenic as predicted by DEREK Nexus.

This study is classified as acceptable for assessment based on methodolgy and documentation. This study satisfy the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) and the data is part of an overall assessment.

Justification for classification or non-classification

Based on the study and the QSAR prediction results a classification according to Regulation (EC) No. 1272/2008 (CLP), Annex I, is not warranted.