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Genetic toxicity in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: modern guideline study, conducted according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
his-operon, trp-operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphthoflavone
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2750, 5500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine
Remarks:
with and without metabolic activation
Details on test system and experimental conditions:
The test substance N4-Amine N,N’-Bis-(3-Aminopropyl)-ethylenediamine was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay both in the standard plate test and in the preincubation test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA.

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1 000; 2 750 and 5 500 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1 000; 2 750 and 5 500 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al.

Preincubation Test
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Evaluation criteria:
The test substance is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at highest test conditions (5500 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Under the experimental conditions chosen, it is concluded that N4-Amine N,N’- Bis-(3-Aminopropyl)-ethylenediamine is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.
Executive summary:

According to the results of the study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

Additional information

in vitro:

Bacterial gene mutation:

The test substance N4-Amine N,N’-Bis-(3-Aminopropyl)-ethylenediamine was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse

mutation assay (OECD 471, GLP, BASF 2012). Following strains were used in this study: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a dose range of 33 μg - 5 500 μg/plate (SPT) and 33 μg - 5 500 μg/plate (PIT), respectively. Standard plate test (SPT) and preincubation test (PIT) both were conducted with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and

test conditions at 5 500 μg/plate. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance N4-Amine N,N’-Bis-(3-Aminopropyl)-ethylenediamine is not mutagenic in the Salmonella

typhimurium/Escherichia coli reverse mutation assay in the absence and the presence ofmetabolic activation.

Mammalian gene mutation:

For this endpoint no study is available for the test substance N3 -Amine. But reliable data is available from N3/N4 -Amine (CAS 68909 -99 -9) which is used as read across.

The study was performed to investigate the potential of N3/N4-Amingemisch to induce gene mutations at the HPRT locus in Ovary (CHO) Cells of the Chinese hamster (OECD 476, GLP, BASF 1997). The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation. The test article was tested at the following concentrations:

Experiment I:

without S9 mix: 30.0; 100.0; 300.0; 1000.0; 3000.0 and 5000.0 µg/mL

with S9 mix: 30.0; 300.0; 1000.0; 3000.0 and 5000.0** µg/mL

Experiment II:

without S9 mix: 30.0; 100.0; 300.0; 1000.0; 2000.0 and 3000.0 µg/mL

with S9 mix: 100.0; 1000.0; 2000.0; and 4000.0 µg/mL

No precipitation of the test article 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 µ/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 expeniments up to the maximal concentration of the test article 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. In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in ovary (CHO) cells of the Chinese hamster. Therefore, N3/N4-Amingemisch is considered to be non-mutagenic in this HPRT assay.

Chromosomal aberration:

For this endpoint no study is available for the test substance N3 -Amine. But reliable data is available from N3/N4 -Amine (CAS 68909 -99 -9) which is used as read across.

The test article N3/N4-Amingemisch, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments (OECD 473, GLP, BASF 1997). The chromosomes were prepared 18 h (exp. I and II) and 28 h (exp. II) after start of treatment with the test article. The treatment period was 4 h with metabolic activation and 18 h as well as 28 h (exp. II only) without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The applicable concentration range of the test article for the cytogenetic experiments was assessed in a pre-test, using the determination of cell numbers 24 h after start of treatment as indicator for toxicity response. The highest concentration used in the pre-test on toxicity (5000 µg/mL) was chosen according to the current OECD Guideline for in vitro mammalian cytogenetic tests. Test article concentrations between 25 and 5000 µg/mL (with and without S9-mix) were chosen for the examination of the cytotoxic potential. In the absence of S9 mix toxic effects indicated by a strong reduction of the cell number (< 50% of control) were observed after treatment with 2500 µg/mL and above, whereas in the presence of S9 mnix toxic effects were observed after treatment with 5000 µg/mL only.

In experiment I, test article concentrations within a range from 125 - 2000 µg/mL (without S9 mix) and 250 - 4000 µg/mL (with S9 mix) were applied for the investigation of the potential to induce cytogenetic damage. In experiment II, the applied concentration ranges were 125 - 1400 µg/mL in the absence of S9 mix or 250 - 4500 µg/mL in the presence of S9 mix. In the absence of S9 mix, in both experiments, the mitotic indices were reduced at interval 18 h after treatment with 500 µg/mL and 1000 µg/mL (exp. II only). In addition, reduced cell numbers were observed at interval 18 h after treatment with 1000 µg/mL and at interval 28 h after treatment with 500 µg/mL. In the presence of S9 mix in experiment II the mitotic indices were reduced in cultures after treatment with 4000 µg/mL (18 h interval) and 4500 µg/mL (28 h interval). In both independent experiments, neither biologically relevant nor statistically significant increases in cells with structural aberrations were observed after treatment with the test article. In both experiments, no biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the solvent controls. Appropriate mutagens were used as positive controls. They induced statistically significant

increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, N3/N4-Amingemisch is considered to be non-mutagenic in this chromosome aberration test.

in vivo:

No data available.

Disregared studies:

There are six studies available concerning bacterial gene mutation (2 Ames tests), mammalian gene mutation (1 HGPRT test), cytogenicity tests (mouse lymphoma assay, sister chromatide exchange assay) and an micronucleus assay in vivo mandated by Aldrich Chem Co Inc. (1991/1992). The test substance used for these assays had a technical grade (95%) with unknown impurities. The reports indicate that the test substance is active in the mouse lymphoma assay, micronucleus assay, sister chromatid exchange assay and HGPRT test. No activity was detected in the Ames test either with or without adjustment of pH. The test substance was adjusted to pH 7.5 with sulfuric acid prior to use in all assays. Thus, the test data are probably test results obtained on the sulfate or bisulfate salts of the N4 Amine. Since polyamines are not implicated as group of materials as known mutagens it is assumed that the positive results are due to unknown impurities. BASF manufactures N4 Amine and N3/N4 Amine (used for read across) by itself and does not use the chemical from another company which might be different in composition therefore these studies were disregarded and not used for classification.


Short description of key information:
in vitro:
Ames: negative (GLP, OECD 471, BASF 2012)
A mammalian gene mutation assay and a cytogenetic mutation assay are not available for the test substance. But these test are available for the N3/N4 amine which contains the test substance (N4 amine) to 45-55% and are used as read across.
HPRT: negative (CAS 68909-99-9, GLP, OECD 476, BASF 1997)
CA: negative (CAS 68909-99-9, GLP, OECD 473, BASF 1997)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the in vitro genetic toxicity studies, the substance does not need to be classified according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.