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Toxicological information

Genetic toxicity: in vitro

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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:
1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed according to OECD test guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
additionally modified version for azo-dyes
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
The assay was performed with and without rat liver and hamster liver microsomal activation.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
TA 1537: hisC3076, rfa, uvrB
TA 98: hisD3052, rfa, uvrB +R
TA 1535: hisG46, rfa, uvrB
TA 100: hisG46, rfa, uvrB +R
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:
S9 mix from rat liver (10%) and S9 mix from hamster liver (30%)
Test concentrations with justification for top dose:
Experiment I: 0, 4, 20, 100, 500, 2500 and 5000 µg/plate;
Experiment II and toxicity test: 0, 4, 20, 100, 500, 2500 and 5000 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolica activation: sodium azide (TA100, TA1535), 9-aminoacridine (TA1537), 2-nitrofluorene (TA98); with rat liver S9-mix: 2-aminoanthracene (all strains); with hamster liver S9-mix: 2-aminoanthracene (TA100, TA1535, TA1537); congo red (TA98)
Details on test system and experimental conditions:
The assay was performed in two independent experiments:

experiment I: plate incorporation assay with and without induced rat liver S9 mix
experiment II: pre-incubation test with and without non-induced hamster liver S9 mix

Two independent experiments for each of the two protocols (incorporation and pre-incubation) were performed.

Hamster liver S9 mix, but not rat liver S9 mix, contained the reductive agent FMN.


DURATION
- Preincubation period: 30 min, 30°C
- Exposure duration: after solidification the plates were incubated for about 48 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more
Evaluation criteria:
A test item is considered as a mutagen if
a) it produces at least a 2-fold increase in the mean number of
revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate control at complete bacterial lawn, or
b) it induces a dose dependent increase in the mean number of revertants per plate of at least
one of the tester strains over the mean number of revertants per plate of the appropriate vehicle
control in at least two or three concentrations of the test compound at complete bacterial background lawn.
Statistics:
not required

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:
no cytotoxicity
Remarks:
Visible precipitation of the test compound was observed at concentrations of 500 µg/plate and higher
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'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the experimental conditions reported, including the Prival test method modification for azo compounds, the test item did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.