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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
Study period:
2000
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Except the selection of the tester strains
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
yes
Remarks:
Except the selection of the tester strains
Qualifier:
according to guideline
Guideline:
other: EEC Directive 2000/32, L 136, Annex 4D, B13/14, dated June 08, 2000
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Etilefrine
EC Number:
211-910-4
EC Name:
Etilefrine
Cas Number:
709-55-7
Molecular formula:
C10H15NO2
IUPAC Name:
3-[2-(ethylamino)-1-hydroxyethyl]phenol
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name : Effortil-Rohbase
Batch No.: 310
Aggregate State at RT: solid
Colour: white
Purity: >98%
Analysis: meldting point
Certificate of Analysis: 19.07.2000
Sorage: in original container, at room temperature

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: his G46;rfa;uvrB;R-factor: base-pair substitutions
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: his D3052; rfa; uvrB;R-factor: frame shift mutations
Metabolic activation:
with and without
Test concentrations with justification for top dose:
31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0ug/plate
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-Nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)
Remarks:
The stability of the positive control substances in solution is unknown but a mutagenic response in the expected range is sufficient evidence of biological stability.
Details on test system and experimental conditions:
For the pre-incubation method 100 ul of the test item solution was preincubated with the tester strains (100ul) and sterile buffer or the metabolic activation system (500ul) for 60 minutes at 37°C prior to adding the overlay agar (2000ul) and pouring onto the surface of a minimal agar plate.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37°C for at least 48h in the dark.
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation).

For positive controls solved in water:
due to a lack in the evaluation-software the mutation factor is correlated to the solvent control and not to the negative (water) control. This does not influence the validity of the data.

A test is considered acceptable if for each strain:
the bacteria demonstrate their typical responses to crystal violet and ampicillin
the control plates without S9 mix are within the following ranges (range of spontaneous reversion frequencies, historical control data range):
TA 98: 18-63
TA 100: 79-197
corresponding background growth an both negative control and test plates is observed.
the positive controls show a distinct enhancement over the control plate.

A test item is considered as mutagenic if:
- a dose-related increase in the number of revertants occurs and/or
- a reproducible biologically relevant positive response for at least one of the test points occurs
in at least one strain with or without metabolic activation.
According to the new OECD guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.
A biologically relevant increase is described as follows:
if in test strain TA 100 the number of reversions is at least twice as high
if in test strain TA 98 the number of reversions is at least three times higher
as compared to the spontaneous reversion rate.

Results and discussion

Test resultsopen allclose all
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 specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Executive summary:

In order to investigate the potential of Effortil-Rohbase for its ability toinduce gene mutations the plate incorporation test (experiment I) and the pre­incubation test (experiment II) were perfoiuied with theSalmonellatyphimuriumstrains TA 98 and TA 100.

The test item was tested in two independent experiments at severalconcentrations. Each assay was conducted with and without metabolicactivation (S9 mix). The concentrations, including the controls, were tested intriplicate. The following concentrations of the test item were prepared andused in the experiments:

31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg/plate

Slight toxic effects indicated by a reduction of the background lawn of thetest item were observed in experiment II with metabolic activation with bothtester strains at 2500.0 and 5000.0 ug/plate. No toxic effects could beobserved in the first experiment and in the second experiment without metabolicactivation up to the highest investigated dose in both strains used.

No substantial increases in the revertant colony numbers of any of the twotest strains were detected at any dose level of the test item either with orwithout metabolic activation in both independently performed experiments.

As positive controls reference mutagens were tested in parallel to the testitem. They showed a distinct increase of induced revertant colonies