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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two Ames bacterial reverse mutation assays available, and in addition an in-vitro mammalian cell gene mutation assay, the (L5178 TK+/-) mouse lymphoma assay and a human lymphocyte chromosomal aberration assay.  All studies are Klimisch 1 and all are negative.

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:
11. May 1997 - 01. Jul 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (OECD 471/472)
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
; OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidin operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from Aroclor 1254 treated male Sprague-Dawley rats.
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S-9 mix: all strains 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 4-nitro-o-phenylendiamine; TA1537: 9-aminoacridine; E.coli WP2 uvrA: N-ethyl-N'-nitro-N-nitrosoguanidine.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h


NUMBER OF REPLICATIONS: 3

METHOD OF APPLICATION: standard plate test

DURATION
- Exposure duration: 48 - 72 h


NUMBER OF REPLICATIONS: 3

Evaluation criteria:
The test chemical 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 S-9 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 two experiments carried out independently of each other.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Complete solubility of test substance in aqua dest.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Standart plate test:

 Dose (µg/plate)  TA1535     TA100     TA1537     TA98        E. coli WP2 uvrA            
   -S9  +S9  -S9  +S9  -S9  +S9 -S9  +S9 -S9  +S9             
 0 20±2 20±1 153±17 158±9 13±4 12±3 31±3 45±3 37±7 36±5            
20  18±1 17±1 147±12 161±9 13±2 12±2 34±3 42±1 31±1 26±3            
 100 17±1 16±1 163±5 157±12 11±1 16±2 32±2 37±2 34±4 30±2            
 500 14±3 20±1 153±7 157±16 11±2 14±2 34±3 38±6 34±3 32±3            
 2500 15±2 17±2 152±14 156±5 13±4 14±2 32±3 42±3 28±3 31±3            
 5000 14±2 17±3 154±6 163±5 11±2 12±1 33±2 40±2 29±1 34±7            
 2-AA - 149±7 - 1474±62  - 179±2 1237±32  - 317±37            
 MNNG  955±36 - 858±42 - -  - -  - - -            
 AAC - - - - 750±6 - - - - -            
 NPD  - - - - - - 1104±77 -            
 ENNG  - - - - - - - - 1185±189 -            

Mean ± SD

Preincubation-test:

 Dose (µg/plate)  TA1535     TA100     TA1537     TA98     E. coli WP2 uvrA                 
   -S9  +S9  -S9  +S9  -S9  +S9 -S9  +S9  -S9 +S9               
 0 20±2 20±2 129±2 124±19 11±2  11±2 30±1 41±3 29±1 35±3              
20  21±3 17±1 146±16 151±21 8±2  10±1 26±7 39±6 25±3 29±4              
 100 17±1 17±2 143±11 195±18 10±1  10±1 25±3 41±6 25±6 30±4              
 500 17±3 18±1 150±20 153±7 9±1  9±1 21±1 30±10 26±3 32±3              
 2500 17±1 16±2 158±13 176±25 8±2  9±1 23±4 41±1 24±2 28±3              
 5000 12±1 14±3 130±4 173±25 7±2  7±2 20±1 35±6 19±8 33±3              
 2-AA  -  154±22 - 805±107 -  148±10 - 1173±41 - 242±8              
 MNNG 981±45 - 1232±70 - 971±34 - - - -  -              
 AAC  - - - - - - -  -              
 NPD  - - - - -  - 1228±98 -  -              
ENNG - - - - - - - - 875±45 -              

Mean ± SD

2-AA: 2-aminoanthracene;

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

ENNG; N-ethyl-N-nitro-N-nitrosoguanidine

NPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

Under the conditions tested Diethylaminopropylamin is non mutagenic in the bacterial AMES-test with or without metabolic activation.

The positive control gave the expected values.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

There are two Ames bacterial reverse mutation assays available; the key study is BASFAG40M0641/964385 Genetic Toxicity in vitro. Ames test from 1997 which is a Klimisch 1 study carried out to GLP and OECD guideline 471, this study included four strains of S. typhimurium, TA1535, TA1537, TA98 and TA100 and E. Coli WP2 uvrA. The study was negative with and without metabolic activation. The second Ames test which is also Klimisch 1, 109-01-3 Genetic toxicity Ames test, was also negative, this study is valid using the same S. typhimurium strans and in addition strain TA1538 but it does not contain either S. typhimurium TA102 or E. Coli WP2 uvr A which are required if certain oxidizing mutagens, cross linking agents and hydrazines are to be detected, the E. Coli WP2 uvr A is included in the BASF study so this aspect is covered. This study was also negative with and without the presence of S9 metabolic activation.

1-methyl piperazine was also negative in the Mouse Lymphoma L5178Y TK +/- assay, 109-01-3 Genetic toxicity in vitro mouse lymphoma 2012, where no toxicologically significant dose-related increases in the mutant frequency were seen at any dose level, either with or without metabolic activation. 

It was also negative for clastogenicity in the human lymphocyte chromosomal aberration study, 109-01-3 Genetic toxicity in vitro chromosomal aberration 2012, where it was found to be moderately toxic. In this study the 1-methyl piperazine did not induce any significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced some slight mitotic inhibition at the 10mM concentration. 1-methyl piperazine was non-clastogenic in human lymphocytes.

All three aspects of in vitro genetic toxicity testing are available covering gene mutation in bacterial and mammalian cells and clastogenicity in human lymphocytes. In the absence of any adverse effects it is clear that 1-methyl piperazine is not genotoxic and there are no effects in vitro requiring clarifications with in vivo genetic toxicology testing.

Justification for selection of genetic toxicity endpoint

For the genetic toxicology end point it is difficult to select a single key study as there are three types of study, the Ames bacterial reverse mutation assay, which investigates the potential for mutations to DNA in bacteria, two of these are available.  There is also an in-vitro mammalian cell gene mutation assay, the (L5178 TK+/-) mouse lymphoma assay.  In addition there is a chromosomal aberration assay in human lymphocytes.  The key Ames test study has been selected for the end point, but the others are all relevant.

Justification for classification or non-classification

There are two Ames, bacterial reverse mutation studies, a Mouse lymphoma L5178Y (TK+/-) mammalian cell mutation assay and an in vitro chromosomal aberration assay in human lymphocytes. All these assays are Klimisch 1 for validity and were conducted to GLP and OECD guidelines.

These assays showed no evidence of mutagenicity or clastogenicty for 1-methyl piperazine, CAS No 109-01-3, therefore there is no data to indicate any requirement for classification for genetic toxicity under the EU CLP (GHS) criteria.