Registration Dossier

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:
09/10 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out in accordance to Directive 92/69/EEC, Part B.13 and B.14, 1992, and the ninth amendment of OECD Guideline for Testing of Chemicals No. 471, 1997, with no deviations reported.

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Jugend, Familie und Gesundheit
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Actinol
- Physical state: crystalline white powder
- Analytical purity: 99.5 %
- Lot/batch No.: 410016
- Expiration date of the lot/batch: 31 July 2000
- Storage condition of test material: at room temperature, protected from sun light
- Other:

Method

Target gene:
pKm 101
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
no applicable
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
concurrent untreated and solvent
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA 1535, TA100 (10 ug/plate)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
TA 1537 (50 ug/plate), TA 98 (10 ug/plate)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA (5 uL/plate)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA 1535, TA 1537, TA 98, TA 100 (2.5 ug/plate), WP2 uvrA (10 ug/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation): In the test, 100 uL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control), 500 uL S9 mix (for metabolic activation) or S9 mix substitution buffer (for test without metabolic activation), 100 uL bacterial suspension, and 2000 uL overlay agar were poured onto the minimal agar plates.

METHOD OF APPLICATION: preincubation: In the test, 100 uL test solution, 500 uL S9 mix or S9 mix substitution buffer and 100 uL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation, 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on minimal agar plates.
After solidification, the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level including the Controls, three plates were used.
Evaluation criteria:
A test item is considered positive if either a dose related and reproducible increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test item producing neither a reproducible and dose related increase in the number of revertants, nor a biologically relevant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system.
A mutagenic response is described as follows:
A test item is considered mutagenic if in the strains TA 98, TA 100, and WP2 uvrA the number of reversions will be at least twice as high and in the strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate (3,4). Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the
highest dose induced the above described enhancement factors or not.
Statistics:
No statistical evaluation has been applied.

Results and discussion

Test resultsopen allclose all
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
Vehicle 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
Vehicle 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

During the described mutagenicity test and under the experimental conditions, the test substance Actinol did not induce gene mutations by base pair changes or frameshifts of the strains used.
Executive summary:

A study was performed to investigate the potential of Actinol to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA

100, and the Escherichia coli strain WP2 uvrA in compliance with Directive 92/69/EEC, Part B.13 and B.14, 1992.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 ug/plate

The plates incubated with the test item showed normal background growth up to 5000 pg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Actinol at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation

rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

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

Therefore, Actinol is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.