N-(2-aminoethyl)-1,3-propanediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro: Ames: negative (OECD 471, BASF 1990) A mammalian gene mutation assay and a cytogenetic mutation assay are not available for the test substance. But these tests - and an Ames-test - are available for the source substance N3/N4 amine (which contains the N4 amine to 45-55%) and are used as read-across. AMES: negative (CAS 68909-99-9, GLP, OECD 471 and 472, BASF 1997) HPRT: negative (CAS 68909-99-9, GLP, OECD 476, BASF 1997) CA: negative (CAS 68909-99-9, GLP, OECD 473, BASF 1997) Furthermore, an Ames-test for the source substance N4-Amine N,N’- Bis-(3-Aminopropyl)-ethylenediamine is available as read-across: AMES: negative (CAS 10563-26-5, GLP, OECD 471, BASF 2012)

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:

1990-10-18 - 1990-12-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment; only four strains were tested.

Justification for type of information:

Corresponding to the draft decision (CCH-D-2114618994-36-01/D) the registrant agrees to conduct an OECD 471.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

, only four strains

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix prepared from sprange Dawlay rat livers after Aroclor 1254 activation

Test concentrations with justification for top dose:

0, 20 ,100, 500, 2500, 5000 µg/plate (TA 100, TA 1537, TA 98; SPT)
0, 500, 1000, 5000, 7000 µg/plate (TA 1535; SPT)
0, 4, 20, 100, 500, 2500 µg/plate (all tester strains; PIT)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details

Details on test system and experimental conditions:

Standard plate test
Test tubes containing 2 ml portions of saft agar kept in a water bath at 45°C, and the remaining companents are added in the following order:
0.1 mL test solution
0.1 mL bacterial suspension
0.5 mL S-9 mix (in tests with metabolic activatian) or
0.5 mL phosphate buffer (in tests without metabalic activatian)
After mixing, the samples are poured onto Vogel-Bonner agar plates. After incubation at 37°C for 48 -72 hours in the dark, the bacterial calonies (his+ revertants) are counted.


Preincubation test
0.1 mL test solution or vehicle, 0.1 ml bacterial Suspension and 0.5 mL S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx.30 seconds.

Both Tests
In each experiment 3 Test plates per dose per control used. After incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Posivite Control:
with S-9 mix:
10 µg 2-aminoanthracene (2-AA) (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537and TA 1535

without S-9 mix:
5 µg N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535
10 µg 4-nitro-o-phenylendiamine (NOPD) (dissolved in DMSO) for the strain TA 98
100 µg 9-aminoacridine (AAC) chloride monohydrate (dissolved in DMSO) for the strain TA 1537

The Titer was determined and in regularly measurements the strain characteristics were checked. Sterility control performed.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test hat to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Species / strain:

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

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:

Toxicity: A bacteriotoxic effect was observed in the standard plate test with and without S-9 mix at 5000 µg/plate.
In the preincubation assay bacteriotoxicity was found only without metabolic activation at 2500 µg/plate.
Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here. The slightly enhanced figures in the 1st experiment using TA 1535, which might partly be explained by the very 1ow spontaneous rate was not confirmed in two additional standard plate tests and in a preincubation assay. Therefore, these finding are regarded as incidental and not as an indication of a point mutagenic activity.

The positive controls (+S9-mix: 2AA; -S9-mix: MNNG, NOPD, AAC) showed the expected results.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results in detail (Ames-test, BASF 1990, Project No. 40M0228/894480)

Plate incorporation with S-9 mix (colony number = mean values) - experiment 1:

Dose (µg/per plate)	TA 100	TA 1537	TA 98	TA1535
0 (aqua dest.)	106	11	33	11
20	95	8	34	10
100	96	9	35	13
500	65	11	32	15
2500	72	9	28	26
5000	61	12	21	29
10 µg 2-AA	1563	163	1507	305

Plate incorporation without S-9 mix (colony number = mean values) - experiment 1:

Dose (µg/per plate)	TA 100	TA 1537	TA 98	TA 1535
0 (aqua dest.)	99	6	22	18
20	107	6	22	27
100	112	7	19	25
500	108	8	26	24
2500	95	8	18	23
5000	83	9	-	21
5 µg MNNG	1500			1465
100 µg AAC		539
10 µg NPD			808

Plate incorporation with S-9 mix (colony number = mean values) - experiment 2:

Dose (µg/per plate)	TA 1535
0 (aqua dest.)	18
500	23
1000	22
3000	21
5000	24
7000	19
10 µg AA	195

Plate incorporation without S-9 mix (colony number = mean value) - experiment 2:

Dose (µg/per plate)	TA 1535
0 (aqua dest.)	18
500	27
1000	25
3000	24
5000	23
7000	21
5 µg MNNG	1465

Plate incorporation with S-9 mix (colony number = mean value) - experiment 3:

Dose (µg/per plate)	TA 100	TA 1537	TA 98	TA1535
0 (aqua dest.)	117	15	42	18
4	106	12	45	10
20	120	11	35	12
100	132	11	32	9
500	122	15	24	18
2500	101	10	21	25
10 µg 2-AA	1061	96	687	187

Plate incorporation with S-9 mix (colony number = mean value) - experiment 3:

Dose (µg/per plate)	TA 100	TA 1537	TA 98	TA1535
0 (aqua dest.)	105	12	21	23
4	105	12	14	18
20	120	8	21	19
100	135	12	22	19
500	111	13	21	20
2500	-	2	7	9
5 µg MMNG	981			1010
100 µg AAC		479
10 µg NPD			740

Plate incorporation with S-9 mix (colony number = mean values) - experiment 4:

Dose (µg/per plate)	TA 1535
0 (aqua dest.)	16
500	17
1000	19
3000	24
5000	19
7000	18
10 µg AA	151

Plate incorporation without S-9 mix (colony number = mean value) - experiment 4:

Dose (µg/per plate)	TA 1535
0 (aqua dest.)	14
500	16
1000	19
3000	19
5000	16
7000	10
5 µg MNNG	1777

- : reduced his- background

2-AA: 2-Aminoanthracene (dissolved in DMSO)

MNNG: N-Methyl-N'-nitro-N-nitrosoguanidine (dissolved in DMSO)

AAC: 9-aminoacridine chloride monohydrate (dissolved in DMSO)

NPD: 4-nitro-o-phenylendiamine (dissolved in DMSO)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

in vitro:

 

Bacterial gene mutation, target substance (BASF, 1990)

Toxicity: A bacteriotoxic effect was observed in the standard plate test with and without S-9 mix at 5000 µg/plate. In the preincubation assay bacteriotoxicity was found only without metabolic activation at 2500 µg/plate.

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

According to the results of the present study, the target substance is not mutagenic in the Ames test under the experimental conditions chosen here.

The slightly enhanced figures in the 1st experiment using TA 1535, which might partly be explained by the very low spontaneous rate was not confirmed in two additional standard plate tests and in a preincubation assay. Therefore, these finding are regarded as incidental and not as an indication of a point mutagenic activity.

The positive controls (+S9-mix: 2AA; -S9-mix: MNNG, NOPD, AAC) showed the expected results.

 

Bacterial gene mutation, source substance N3/N4-Amine (BASF, 1997)

Toxicity: A bacteriotoxic effect was observed at doses >= 2,500 µg/plate.

Mutagenicity: An increase in the number of his+ revertants or trp+ was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

According to the results of the present study, the source substance (N3/N4-Amine) is not mutagenic in the Ames test and in the Escherichia coli reverse mutation assay under the experimental conditions chosen here.

 

Chromosome aberration assay, source substance N3/N4-Amine (BASF, 1997)

In both independent experiments, neither biologically relevant nor statistically significant increases in cells with structural aberrations were observed after treatment with the source substance. In both experiments, no biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the source substance (N3/N4-Amine) as compared to the frequencies of the solvent controls. Appropriate mutagens were used as positive controls and showed the expected results.

 

HPRT Test, source substance N3/N4-Amine (BASF, 1997)

The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation.

No precipitation of the source substance occurred up to the maximal concentration neither in the presence nor in the absence of metabolic activation. In the absence of metabolic activation strong toxic effects occurred starting at concentrations of 1000 µg/mL in the first and 2000 µg/mL in the second experiment. In the presence of metabolic activation toxic effects were observed beginning at 3000 µg/mL in the first and at 4000 µg/mL in the second experiment.

No biologically relevant increase in mutant colony numbers was observed in both independent experiments up to the maximal concentration of the source substance neither in the presence nor in the absence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

 

Bacterial gene mutation, source substance N4-Amine (BASF, 2012)

Toxicity: A weakly bacteriotoxic effect was observed only in the preincubation test depending on the test conditions at doses > 2500 µg/plate.

Mutagenicity: An increase in the number of his+ revertants was not observed both in the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.

According to the results of the present study, the source substance is not mutagenic in the Ames test under the experimental conditions chosen here. (This result is supported by a previous peformed study with N4 -amine which is listed for the sake of completeness (BASF, 1990)).

in vivo:

Disregarded study with RA substance due to methological deficiencies.

Justification for selection of genetic toxicity endpoint
Only one study available with the test substance. Estimated Klimisch Rating: 2
Conclusion is supported by read-across approach to two other source substances.

Justification for classification or non-classification

Based on the results of the in vitro genetic toxicity study (Ames test), it is concluded that the target test substance has not to be classified:

 

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation EC (No) 1272/2008. Based on the available study it is concluded that the target test substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008 , as amended for the sixth time in Regulation (EC) No 605/2014.

 

This justification is supported by the results of the in vitro studies of two source substances (read-across approach).