Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline study, not in triplicate (duplicates only)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1997
Deviations:
yes
Remarks:
only plate duplicates per treatment were used
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
Salmonella: his-
Escherichia: trp-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of liver from rats induced with Phenobarbital/ 5,6-benzoflavone
Test concentrations with justification for top dose:
Dose-finding test: 5, 20, 78, 313, 1250 and 5000 µg/ plate
Main test: 313, 625, 1250, 2500 and 5000 µg/ plate
Confirmation test (only with TA 98 and TA100 with metabolic activation): 1000, 2000, 3000, 4000 and 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: see "Details on test system"
Details on test system and experimental conditions:
Dose finding test, main test & confirmation test: Preincubation assay
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S-9 mix (10% S9 supernatant plus 90% cofactor solution) are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 mL of top agar is added and, after mixing, the samples are poured onto the minimum glucose agar plates.

In each experiment 2 test plates per dose or per control used; after incubation at 37°C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

POSITIVE CONTROL:
with metabolic activation: 2-aminoanthracene for TA 1535 (2.0 μg/plate) and WP2 uvr A (10 µg/plate); benzo[a]pyrene (5.0 μg/plate) for TA 98, TA 100 and TA 1537
without metabolic activation: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide for TA 98 (0.1 μg/plate), TA100 and WP2 uvr A(both 0.01 μg/plate); sodium azide (0.5 μg/plate) for TA 1535; 9-aminoacridine (80 μg/plate) for TA 1537.
All substances were dissolved in DMSO.

The titer was determined and the strain characteristics were checked in regular measurements. Sterility control was performed.
Evaluation criteria:
According to the report, the test substance was considered to have a reverse mutagenic potential when the number of revertant colonies per plate treated with the test substance was more than twice that of the negative (vehicle) control and there was reproducibility in test results, and when negative (vehicle) control values, positive control values and test results satisfied the acceptable standards.
Generally, a dose dependency should be observed in addition.
Statistics:
The arithmetic mean was calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
in precipitating doses
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 1535, TA 1537 and TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: TA 98
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Dose-finding test (Preincubation test; 5 - 5000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor* dose dependency Assessment
TA 98 no 19 1.1 no negative
  yes 33 1.1 no negative
TA 100 no 117 1.1 no negative
  yes 138 1.1 no negative
TA 1535 no 8 1.0 no negative
  yes 9 0.9 no negative
TA 1537 no 7 0.9 no negative
  yes 12 0.9 no negative
WP2 uvr A no 19 1.1 no negative
  yes 25 0.9 no negative
Main test (Preincubation test; 313 - 5000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor* dose dependency Assessment
TA 98 no 25 n.a. no n.a.
  yes 36 n.a. no n.a.
TA 100 no 119 n.a. no n.a.
  yes 123 n.a. no n.a.
TA 1535 no 11 n.a. no n.a.
  yes 11 n.a. no n.a.
TA 1537 no 7 n.a. no n.a.
  yes 12 n.a. no n.a.
WP2 uvr A no 27 n.a. no n.a.
  yes 26 n.a. no n.a.
Confirmation test (Preincubation test; 1000 - 5000 µg/plate)
Strain Metabolic activation system mean revertants in Controls maximum revertant factor* dose dependency Assessment
TA 98 no only neg. ctrl. performed
  yes 34 n.a. no n.a.
TA 100 no only neg. ctrl. performed
  yes 134 n.a. no n.a.
* in non-cytotoxic, non-precipitating doses (>= 313 μg/plate without metabolic activation and >= 78 μg/plate with metabolic activation)
n.a. = not applicable

In TA98 with metabolic activation in precipitating doses, the revertant factor was maximum increased by a factor of 5.2, 5.3 and 4.8 in the highest dose in the dose finding, main and confirmation test, respectively; the increase of revertants showed a dose dependence in all three tests. In all other treatments with precipitating doses, no relevant increase of mean revertant number by a factor >= 2.0 was observed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation in precipitating doses (TA 98)