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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:
17.11.1998 - 24.02.1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted in GLP compliance and in accordance with an OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
AG-EE 623 (S)-amine
IUPAC Name:
AG-EE 623 (S)-amine
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): AG-EE 623 (S)-amine
- Physical state: Light yellow viscous
- Purity test date: August 28, 1998
- Lot/batch No.: WB 501
- Expiration date of the lot/batch: August 31, 2000
- Storage condition of test material: At room temperaturein the dark (ambient humidity)
- Other:

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Five concentration Ievels of 1, 3, 10, 30 and 100µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Plate Incorporation:
0.1 ml of a bacterial shaking culture (6 hr, exponential phase), 0.1 ml vehicle or test article solution
and 0.5 ml phosphate buffer or S9-mix were added to 2 ml soft agar. After vortexing, these mixtures
were overlaid immediately onto minimal medium plates in triplicate (control: 6 plates).


Preincubation
The results were confirmed in an independent experiment using the preincubation technique. 0.1 ml
vehicle or test article solution, 0.5 ml phosphate buffer or S9-mix and 0.1 ml of a bacterial culture
were preincubated and shaken for 20 min at 37°C. Then 2 ml soft agar were added to the mixture and
after vortexing overlaid onto minimal medium plate.
All tests were performed in the presence or absence ofrat liver microsomal enzymes. Finally, the titer
of the initial bacterial suspension was determined with appropriated culture plates (5 replicates),
respectively.
Evaluation criteria:
Revertant his + colonies were counted using the automatic colony counter IPI Analyser 982 B after
incubation at 37°C for 2 days (TA 102: 3 days). For all replicate platings, the mean number of
revertants per test concentration was calculated.

The condition of the background bacterial lawn (residual growth by histidine residues) was evaluated
macroscopically for evidence of bacteriotoxicity due to the test article. If extreme thinning or complete
lack of the microcolony lawn compared to the vehicle control plates was observed, no revertants were
counted. Evidence of macroscopic test article precipitate in the agar overlay was recorded.

Evaluation Criteria
A reproducible, concentration-dependent increase in the number of revertants of at least one tester
strain over the vehicle control value andlor outside the historical control range is indicative of
genotoxic activity. Since the results were unequivocal, no detailed statistical evaluationwas
performed.A reproducible, concentration-dependent increase in the number of revertants of at least one tester
strain over the vehicle control value andlor outside the historical control range is indicative of
genotoxic activity. Since the results were unequivocal, no detailed statistical evaluation was
performed.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>= 10µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Solubility and Toxicity:

The test article AG-EE 623 (S)-amine was tested in a concentration range of 1-100 µg/plate. Higher concentrations caused strong toxicity. A weak strain- and concentration-dependent toxicity, as seen by a reduced background lawn and/or a decrease of absolute revertant numbers, was observed at the higher concentrations >= 10 µg/plate in the presence and absence of liver enzymes. The strain S. typhimurium TA 102 was most sensitive.

Mutagenicity:

The mean number of bacteria tested varied between 0.2 and 2.0 x 108/plate AG-EE 623 (S)-amine. In the repeat experiment no titer was determined in the strains S. typhimurium TA 1537 and TA 98.

Control plates (vehicle control) showed spontaneaus revertant colonies for the tester strains of S. typhimurium at frequencies similar to those described in the Iiterature and within the historical control range for our laboratory. The number of revertants was not increased after treatrnent with concentrations ranging from 1 to 100 µg/plate AG-EE 623 (S)-amine in all tester strains (plate technics). Addition of liver homogenates from rats pretreated with Aroclor 1254 had no influence on mutation induction. The negative results were qualitatively confirmed by the repeated experiment using preincubation. In the nonactivation system, however, the number of spontaneaus revertants for S. typhimurium TA 1535 exceeded the historical baseline following preincubation. Due to the fact that the positive control showed the known response the assay is regarded valid and no repetition was clone.

Positive Controls:

The diagnostic mutagen NaN3, 9-AA, 2-NF and MMC in the absence and 2-AA in the presence of mammalian liver enzymes showed the expected strain specific responses. The results with the indirect mutagen confirmed the metabolic activation capacity of the S9 fractions used.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on these results it is concluded, that AG-EE 623 (S)-amine, when tested up to toxic
concentrations, caused neither base-pair substitutions nor frameshift mutations in bacteria. No
evidence of genotoxic activity was observed in different strains of S. typhimurium in the absence and
presence of metabolic activation when tested to the limits of toxicity. The test compound is, therefore,
classified as "Ames negative".