Ethanesulfonic acid, 1-(dimethylamino)-2-hydroxy-, sodium salt (1:1)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 17 - 19, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Guideline:

other: AMES II

Deviations:

not specified

Principles of method if other than guideline:

The chemical intermediate PD 159 NA of the BIBW 2992 MA2 synthesis was investigated
in a modified bacterial reverse mutation test as described by Ames et al. (1975). The
traditional Salmonella microsome mutation assay (Ames test) is used extensively as a
routine test for mutagenicity for more than 30 years. The high throughput microtitre-based
version, called Ames II, is based on the same genetic principle (base-pair substitution and
frameshift mutations in the his operon of S. typhimurium) as the traditional Ames test but
combined with the fluctuation method (Gee et al., 1998).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

sodium 2-(dimethylamino)-1-hydroxyethane-1-sulfonate

EC Number:

929-207-2

Cas Number:

1172126-47-4

Molecular formula:

C4 H11 Cl N Na O4 S

IUPAC Name:

sodium 2-(dimethylamino)-1-hydroxyethane-1-sulfonate

Method

Species / strain

Species / strain 1

Species / strain / cell type:

other: S. typhimurium TA Mix (TA 7001-7006)

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

PD 159 NA was dissolved in dimethylsulfoxide DMSO (negative control). Therefore, 7
concentration levels from 1 to 5000 μg/mL were tested along with the appropriate negative
and positive controls.

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

other: 2-aminoanthracene

Details on test system and experimental conditions:

The assay was performed according to the instruction manual for the Ames II
(Xenometrix, Boulder/USA). Vehicle, test substance or positive control in a volume of
0.01 mL were incubated with 0.24 mL bacterial overnight culture (ca 107/mL) in exposure
medium in 24-well plates for 90 min at 37°C and 250 rpm. With metabolic activation
0.2 mL strain mixture and 0.04 mL S9-mix (30%) were used.
After 90 min the exposed cultures were diluted with pH indicator medium lacking histidine
and aliquoted into 48 wells of a 384-well plate (3 replicates) using an 8-channel pipettor.
The plates were incubated for 48 h at 37°C.
To confirm the sensitivity of the tester strains and the metabolic capacity of the S9
fractions, the diagnostic mutagens 2-nitrofluorene (2-NF), 4-nitroquinoline-N-oxide
(4-NQO) and 2-aminoanthracene (2-AA) were used, respectively.

Rationale for test conditions:

The experiment is regarded valid, if the vehicle control showed the normal spontaneous
revertant frequency and the diagnostic mutagens caused the expected increase in the
mutation rate.

Evaluation criteria:

The individual test chemicals were classified according to the following criteria:
Negative: ≤8/48 wells Equivocal: 9-12/48 wells Positive: ≥13/48 wells

Statistics:

The pH indicator bromocresol purple turns the colour of the cultures from blue to yellow
as the pH drops due to the accumulation of catabolites from the metabolic activity of
revertant cells. The number of positive wells (yellow) out of a total of 48 wells is an
indication of the frequency of reversion per replicate per dose and was compared to the
number of spontaneous revertant wells of the solvent control. Each test point contains
48 wells of a 384-well plate. In each 48-well section, the wells were scored for the number
of revertant wells (yellow) and the mean value of the triplicates was calculated.

Results and discussion

Test results

Test results 1

Species / strain:

other: S. typhimurium TA Mix (TA 7001-7006)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Based on the described results it is concluded, that PD 159 NA, when tested up to
recommended concentrations, caused neither base-pair substitutions nor frameshift
mutations in bacteria. No evidence of genotoxic activity was observed in a series of
S. typhimurium tester strains (TA Mix and TA 98) in the absence and presence of
metabolic activation. Therefore, the test article can be classified as "Ames II
negative".