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Diss Factsheets

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:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD 471 and EEC Directive 84/449, B. 14. Study conducted under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Veratrylcyanid, krist. SF
IUPAC Name:
Veratrylcyanid, krist. SF
Constituent 2
Reference substance name:
91/82
IUPAC Name:
91/82
Details on test material:
- name of the test substance: Veratrylcyanid, krist. SF
- Batch No.: 26530
- Date of manufacturing: August 1990
- Test substance number: 91/82
- Degree of purity: 99.6%
- Appearance, consistency: white crystals
- Storage: room temperature (protected from light)

Method

Target gene:
his
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:
Aroclor induced rat liver S-9 mix.
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine chloride monohydrate
Details on test system and experimental conditions:
Standard plate test

The experimental procedure is based on the method of Ames et al . (1, 2) .

Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0 .6% agar + 0 .6% NaCI) and 10 ml amino acid solution ( minimal amino acid solution for the determination of mutants : 0 .5 mM histidine + 0 .5 mM biotin) are kept in a water bath at 45°C, and the remaining
components are added in the following order: 0 .1 ml test solution or solvent 0 .1 ml bacterial suspension 0 .5 ml S-9 mix (in tests with metabolic activation) or 0 .5 ml phosphate buffer (in tests without metabolic activation).

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. ~ 30 seconds .


Preincubation test

The experimental procedure is based on the method described by Yahagi et al . (5) and Matsushima et al . (6) .
0 .1 ml test solution or solvent, 0 .1 ml bacterial suspension and 0 .5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.


Composition of the minimal glucose agar:

980 ml aqua dest .
20 ml Vogel-Bonner E medium
15 g Difco bacto aga r
20 g D-glucose, monohydrate .
After incubation at 37°C for 48 hours in the dark, the bacterial colonies ( his revertants) are counted.

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 (control)
- dose-response relationship
- reproducibility of the results.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: >= 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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

An increase in the number of his revertants was not observed both in the standard plate test and in the pre-incubation test either without S-9 mix or after the addition of a metabolizing system.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

According to the results of the present study, the test substance Veratrylcyanid is not mutagenic in the Ames test under the experimental conditions chosen here. Thus, no classification is warranted according to Regulation (EU) No. 1272/2008 and Directive 67/548/EEC.