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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No Escherichia coli or S. typhimurium TA102 included
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-aminoethylamino)ethanol
EC Number:
203-867-5
EC Name:
2-(2-aminoethylamino)ethanol
Cas Number:
111-41-1
Molecular formula:
C4H12N2O
IUPAC Name:
2-[(2-aminoethyl)amino]ethanol
Test material form:
not specified

Method

Target gene:
- S.typhimurium: Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : male Sprague-Dawley rats (200 - 300 g) received a single intraperitoneal injection of 500 mg Aroclor 1254 (as a 20% solution in peanut oil - w/v) per kg body weight.
- method of preparation of S9 mix : The S-9 mix is prepared freshly prior to each experiment. For this purpose, a sufficient amount of S-9 fraction is thawed at room temperature and 3 volumes of S-9 fraction are mixed with 7 volumes of S-9 supplement (cofactors).
Test concentrations with justification for top dose:
Experiment 1: (Standard plate test with and without S-9 mix Strains: TA1535, TA 100 . TA 1537, TA 98)
Doses: 20, 100, 500, 2500 and 5000 μg/plate

Experiment 2: (Standard plate test with and without S-9 mix Strains: TA1535, TA100, TA1537, TA98)
Doses: 2000, 4000, 6000 and 8000 μg/plate
Number of plates: 3 test plates per dose or control.

Experiment 3: (preincubation test with and without S-9 mix. Strains: TA1535, TA100, TA1537, TA98)
Doses: 20, 100, 500, 2500, 5000 μg/plate

Experiment 4: (preincubation test with and without S-9 mix. Strains: TA1535)
Doses: 20, 100, 500, 2500, 5000 μg/plate
Vehicle / solvent:
- Vehicle/solvent used: No solvent was used, the test substance was competely dissolved in aqua dest .
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S-9: 5 μg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO ) for the strains TA 100 and TA 1535
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Remarks:
without S-9: 10 μg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S-9: 100 μg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S-9: 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two standard plate tests and two preincubation tests

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: a germ density of ≥ 10*E8 bacteria/mL
- Test substance added in agar (plate incorporation)

TREATMENT (Experiment 1 and 2, standard plate test)
- Preincubation period: no preincubation
- Exposure duration: 48 hours in the dark
- Exposure temperature: 37°C

TREATMENT (Experiment 3 and 4, preincubation test)
- Preincubation period: 20 minutes preincubation at 37°C
- Exposure duration: 48 hours in the dark
- Exposure temperature: 37 °C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The reduction of the bacterial background growth, and decreasse in the number of his+ revertants.
Evaluation criteria:
Evaluation criteria
In general, a substance to be characterized as positive in the Ames test has to fulfill the following
requirements:
- doubling of the spontaneous mutation rate ( compared to the control)
- dose-response relationship
- reproducibility of the results
Statistics:
No statistical analysis was performed.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only in the preincubated test reduced his- growth at doses ≥2500 µg/plate.
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only in the preincubated test reduced his- growth at doses ≥5000 µg/plate.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only in the preincubated test reduced his- growth at doses ≥2500 µg/plate.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only in the preincubated test reduced his- growth at doses ≥5000 µg/plate.
Additional information on results:
Ames test:
- Signs of toxicity : A bacteriotoxic effect (reduced his background growth, decrease in the number of his+ revertants) was observed only in the preincubation test depending on the strain at doses > 2500 µg/plate.
- Individual plate counts: See attached files
- Mean number of revertant colonies per plate and standard deviation:See attached files

Applicant's summary and conclusion

Conclusions:
An Ames study was performed with TA 1535, TA1537, TA98 and TA100 following OECD guideline 471, and showed that the test substance Aminoethylethanolamin is not mutagenic in the Ames test under the experimental conditions used.
Executive summary:

An in vitro gene mutation (AMES) test was perfored similar to OECD 471 guideline with strains TA 1535, TA1537, TA98 and TA 100. All bacteria strains showed negative response up to 8000 ug/plate in the standard plate test and up to 5000 ug/plate in the preincubated test, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was obeserved. Toxicity was observed only in the preincubation test depending on the strain at doses ≥ 2500 ug/plate.

Based on the results of this study it is concluded that Aminoethylethanolamin is not mutagenic in the Salmonella typhimurium reverse mutation assay with or without metabolic activation.

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