Registration Dossier

Administrative data

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: Guideline study conducted according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
yes
Remarks:
2-AA was used as sole indicator for efficacy of the S9 mix
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
(2-hydroxyethyl)(3-hydroxypropyl)dimethylammonium chloride
EC Number:
278-860-3
EC Name:
(2-hydroxyethyl)(3-hydroxypropyl)dimethylammonium chloride
Cas Number:
78182-00-0
Molecular formula:
C7H18NO2.Cl
IUPAC Name:
(2-hydroxyethyl)(3-hydroxypropyl)dimethylazanium chloride
Details on test material:
- Name of test material (as cited in study report): DMAC trocken flüssig
- Physical state: yellowish crystals
- Analytical purity: > 98.8%
- Lot/batch No.: P55
- Storage condition of test material: room temperature

Method

Target gene:
his- and trp-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: The Salmonella strains are checked regularly for deep rough character (rfa) ; UV sensitivity (uvrB) ; ampicillin resistance (R factor plasmid).
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: E . coli WP2 uvrA is checked regulary for UV sensitivity.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9 mix
Test concentrations with justification for top dose:
First experiment (standard plate test SPT): 0; 20; 100; 500; 2500 and 5000 µg/plate
Second experiment (princubation test PIT): 0; 20; 100; 500; 2500 and 5000 µg/plate
Vehicle / solvent:
water
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(sterility control)
Negative solvent / vehicle controls:
yes
Remarks:
(water)
True negative controls:
no
Positive controls:
yes
Remarks:
with S9 mix
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
2.5 µg/plate (DMSO) for TA 1535, TA 100, TA 1537, TA 98; 60 µg/plate in DMSO for Escherichia coli WP2 uvrA
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Remarks:
5 µg/plate (DMSO) for TA 1535 and TA 100
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
10 µg/plate (DMSO) for TA 98
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
9-aminoacridine
Remarks:
100 µg/plate (DMSO) for TA 1537
Positive controls:
yes
Remarks:
without S9 mix
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
5 µg/plate (DMSO) for E. coli WP2 uvrA
Details on test system and experimental conditions:
TESTING
Standard plate test with and without S-9 mix [3 test plates per dose or per control]:
The experimental procedure was based on Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron & Ames (Mut. Res. 113: 173-215, 1983).
Test tubes containing 2 mL portions of soft agar [100 mL agar (0.6% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin for S. typhimurium or 0.5 mM tryptophan for E.coli)] were kept in a water bath at 45°C. 0.1 mL test solution or vehicle (negative control), 0.1 mL fresh bacterial culture and 0.5 mL S9 mix (in case of metabolic activation) or 0.5 mL phosphate buffer (in case of no metabolic activation) were added.
After mixing, the samples were poured onto agar plates and incubated at 37°C for 48 to 72 hours in the dark. After incubation, the bacterial colonies were counted for revertants.

Preincubation test with and without S-9 mix [3 test plates per dose or per control]
The experimental procedure was based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980). 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (in case of metabolic activation) or phosphate buffer (in case of no metabolic activation) were incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates and incubated at 37°C for 48 to 72 hours in the dark. After incubation, the bacterial colonies were counted for revertants.

Titer determination:
In the standard plate test, 0 .1 mL of the overnight cultures was diluted to 10x E-6 in each case. Test tubes containing 2-ml portions of soft agar with amino acid solution as already described were kept in a water bath at 45°C, and 0 .1 mL vehicle (without and with test substance), 0 .1 mL fresh bacterial culture as well as 0 .5 mL S-9 mix were added.
In the preincubation test, 0 .1 mL of the overnight cultures was diluted to 10xE-6 in each case. 0 .1 mL vehicle (with and without test substance), 0 .1 mL bacterial suspension and 0 .5 mL S-9 mix were incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination was added. After mixing, the samples were poured onto the agar plates and incubated at 37°C for 48 to 72 hours in the dark. The bacterial colonies were then counted.

PARAMETERS EVALUATED
Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
Titer:
The titer was generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
Cytotoxicity:
Toxicity was detected by (1) decrease in the number of revertants, (2) clearing or diminution of the background lawn (= reduced his- or trp- background growth) and (3) reduction in the titer. Cytotoxicity was recorded for all test groups both with and without S9 mix in all experiments.
Solubility:
Precipitation of the test item was recorded and indicated. As long as precipitation does not interfere with colony scoring, 5000 µg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities.
Evaluation criteria:
Acceptance criteria:
The experiment is to be considered valid if the following criteria are met :
• The number of revertant colonies in the negative controls is within the normal range of the historical control data for each tester strain;
• The sterility controls reveale no indication of bacterial contamination;
• The positive controls both with and without S-9 mix induce a significant increase in the number of revertant colonies within the range of the historical control data;
• The titer of viable bacteria is > 10xE+9/mL.

Evaluation criteria
The test item is considered positive if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies [about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or with S9 mix];
The test item is considered negative if:
• The number of revertants for all tester strains is within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
for none of the strains tested with and without S9 mix increase in revertant colonies was observed.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
a slight decrease in the number of revertants was occasionally observed at doses >= 2500 µg/plate
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
Remarks:
no increase in revertant colonies was observed either with or without S9 mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
a slight decrease in the number of revertants was occasionally observed at doses >= 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: SPT and PIT
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative