Reaction mass of 3-[(2-aminoethyl)ammonio]propane-1-sulfonate and 3,3'-(ethane-1,2-diyldiammonio)dipropane-1-sulfonate and ethane-1,2-diamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Actual guideline method. GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

3-[(2-aminoethyl)amino]propane-1-sulfonic acid

Cas Number:

14235-54-2

Molecular formula:

C5H14N2O3S

IUPAC Name:

3-[(2-aminoethyl)amino]propane-1-sulfonic acid

Details on test material:

- Physical state: liquid
- Composition of test material, percentage of components:
48.5 % (w/w) 3-[(2-aminoethyl)ammonio]propane-1-sulfonate
51.5 % water
- Lot/batch No.: 06060423/101
- Stability under test conditions: stable
- Storage condition of test material: room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: 79 % RPMI 1640 with L-glutamine, 20 % Fetal Bovine Serum (heat inactivated), 1 % Penicillin-Streptomycin (10000 µg/mL Penicillin, 10000 µg/mL Streptomycin)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0.067, 0.202, 0.607, 1.822 mg/mL
1.822 mg/mL as the highest test substance concentration is 0.01M and was chosen in accordance with the EC directive and the OECD guideline.

Vehicle / solvent:

- Vehicle/solvent used: RPMI1640 for 3 hours treatment, medium for 20 hours treatment

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

RPMI1640 for 3 hours treatment, medium for 20 hours treatment

Negative solvent / vehicle controls:

yes

Remarks:

RPMI1640 for 3 hours treatment, medium for 20 hours treatment

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

controls-treatment without metabolic activation system

Migrated to IUCLID6: treatment without metabolic activation system

Controls 2

Untreated negative controls:

yes

Remarks:

RPMI1640 for 3 hours treatment

Negative solvent / vehicle controls:

yes

Remarks:

RPMI1640 for 3 hours treatment

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

controls-treatment with metabolic activation system

Migrated to IUCLID6: treatment with metabolic activation system

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 h
- Exposure duration: with and without metabolism: 3 h, without metabolism: 20 h
- Further culture period: 15 h
- Colcemid treatment: 2 h
STAIN (for cytogenetic assays): Giemsa solution (10 % v/v in a buffer of 0.067 M KH2PO4 and 0.067 M Na2HPO4 x 2 H2O in deionised water)
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: In general, apart from cultures with very few metaphases or with obviously high numbers of metaphases with aberrations, 100 metaphases per culture were analysed for structural and numerical chromosomal aberrations.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis. This number was then expressed as percentage of mitotic lymphocytes. In cultures where no mitoses were noted at all, the number of lymphocytes was not evaluated.

Evaluation criteria:

• the number of metaphases with numerical aberrations (< 46 chromosomes, > 46 chromosomes, tetraploidy, endoreduplication)
• the number of metaphases with structural aberrations, excluding gaps
• the number of chromatid-type aberrations, excluding gaps
• the number of chromosome-type aberrations, excluding gaps
• the number of gaps (chromatid- and isochromatid-type).

Statistics:

Chi2-Test or Fisher’s Exact Test

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

20 h treatment, in the highest and second highest concentration (1,822 and 0,607 mg/mL)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
All relevant figures were within the range of historical negative controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No marked cytotoxicity (reduction of mitotic indices by more than 50 %, compared to concurrent negative controls) was noted at any of the concentrations tested. The highest tested concentration was 0.01M and thus in accordance with the EC directive and the OECD guideline.

Any other information on results incl. tables

Cytotoxicity

No marked cytotoxicity was noted in any experiment.

Experiment without a metabolic activation system, 3 hours of incubation:

test substance concentration in mg/mL	0,067	0,202	0,607	1,822
mitotic index (% of respective negative control)	100,0	93,8	106,8	80,2

Experiment without a metabolic activation system, 20 hours of incubation:

test substance concentration in mg/mL	0,067	0,202	0,607	1,822
mitotic index (% of respective negative control)	138,8	136,0	125,9	178,4

Experiments with a metabolic activation system, 3 hours of incubation:

test substance concentration in mg/mL	0,067	0,202	0,607	0,607	1,822	1,822
mitotic index (% of respective negative control)	79,2	73,9	71,4	136,9	71,1	116,2

Numerical aberrations

In experiment B without a metabolic activation system and a treatment length of 20 hours the number of metaphases with less than 46 chromosomes was statistically significantly lower at the low test substance concentration of 0.202 mg/mL compared to the concurrent negative controls.

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any other experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Structural aberrations

In experiment B without a metabolic activation system and a treatment length of 20 hours the number of metaphases with structural aberrations was statistically significantly higher at test substance concentrations of 0.607 and 1.822 mg/mL than in the corresponding negative controls (12/200 and 11/200 versus 2/200). The figures were also clearly beyond the data of historical negative controls. No such increase was noted at the lowest test concentration analysed (0.202 mg/mL, 4/200) and there was no clear concentration-response relationship.

In the same experiment uncommon types of aberrations were seen: two pulverisations of chromosomes at 1.822 mg/mL and one centric ring at the low concentration of 0.202 mg/mL.

No marked or statistically significant increases in the number of metaphases with structural aberrations were noted in any other experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not. All figures were within the range of historical negative controls.

Gaps

In experiment B without a metabolic activation system and a treatment length of 20 hours the number of gaps was statistically significantly higher at test substance concentrations of 0.607 and 1.822 mg/mL than in the corresponding negative controls (11/200 and 9/200 versus 0/200). The figures were also clearly beyond the data of historical negative controls. No such increase was noted at the lowest test concentration analysed (0.202 mg/mL, 2/200) and there was no clear concentration-response relationship.

No other statistically significant increases in the number of gaps were noted in any experiment performed at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Positive controls

The positive control substances caused clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive without metabolic activation 20 h treatment, in the highest and second highest concentration

The overall results of the study indicate clastogenic properties of the test substance at the extended treatment length of 20 hours and without the use of a metabolic activation system.

Executive summary:

"3-[(2-aminoethyl)ammonio]propane-1-sulfonate"did not cause marked cytotoxic effects (reduction of mitotic indices by more than 50 %, compared to concurrent negative controls) at any of the concentrations tested. The highest tested concentration was 0.01M and thus in accordance with the EC directive and the OECD guideline.

Statistically significantly higher numbers of metaphases with less than 46 chromosomes at the low test substance concentration of 0.202 mg/mL in one experiment are regarded as a random event without biological relevance.

There was, under the conditions of this study, relevant evidence that"3-[(2-aminoethyl)ammonio]propane-1-sulfonate"did induce structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 20 hours and in the absence of a metabolic activation system, although there was no clear concentration-response relationship. The conclusion is mainly based on a statistically significant increase of metaphases with structural aberrations at the highest and the second highest test substance concentration (1.822 and 0.607 mg/mL) and on the fact that these figures were far beyond the range of historical negative controls. An additional indication for clastogenic effects in this single experiment is the scattered occurence of rare types of structural aberrations (pulverisations and a centric ring), also at the low test substance concentration (0.202 mg/mL). Gaps are not necessarily an indicator for clastogenic effects, but at the highest and the second highest test substance concentration the number of gaps was also increased.

No marked or statistically significant increase in the number of metaphases with aberrations was noted in any other experiment, neither with nor without a metabolic activation system. All relevant figures were within the range of historical negative controls.

The overall results of the study indicate clastogenic properties of the test substance at the extended treatment length of 20 hours and without the use of a metabolic activation system.