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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31.07. - 21.09.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
yes
Remarks:
without impact (see Overall remarks)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report): Semi Dry Absorption (SDA) Product
- Molecular formula (if other than submission substance): not available
- Molecular weight (if other than submission substance): not available

Composition of test substance:
Calcium sulphate 6.49 %
Calcium sulphite 41.40 %
Calcium carbonate 29.50 %
Calcium hydroxide 2.27 %
Calcium chloride 17.98 %
Calcium fluoride 0.57 %
Oxides (SiO2 + Al2O3 + Fe2O3 + TiO2) sum 0.97 %
Sum of toxic metals: As, Be, Cd, Co, Cr, Cu, Hg, Mo, Ni, Pb, Sb, Se, Tl, V, Zn sum < 0.1 %

Batch No.: SDA/0609/Sk
Appearance: White solid powder
Stability / Expiration date: 15 years (06/2024)
- Stability under test conditions: stable
- Storage condition of test material: the substance was stored in PE container at room temperature.
- Other: pH 11 approx. (by contact of application form with universal indicator pH strip moistened with water, strip producer Lach-Ner, s.r.o. Neratovice)

Method

Target gene:
gene for synthesis histidine or tryptophan
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine requiring strain
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: tryptophan requiring strain
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
5, 15, 50, 150, 500 μg/plate
For justification for top dose see box Additional information on results.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water for injection, Ardeapharma a.s., Lot No. 0101030309
- Justification for choice of solvent/vehicle: solubility of the test substance
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
100 ul of Semi Dry Absorption (SDA) Product suspension of required concentration, 0.1 ml 16-18h culture of the tester strain, 0.5 ml relevant buffer and 30 or 100 ul of S9 postmitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml top agar (with trace of histidine or tryptophan) kept in a test tube at 45± 3°C.
After shaking the mixture was poured into a minimal glucose agar plate.

The number of revertant colonies on the plate was counted manually or by using an AccuCount 1000 after 48 - 72 h incubation at 37± 1°C.

NUMBER OF REPLICATIONS: 2 series; each with and without metabolic activation, with positive control and solvent control, triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: total growth
Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, its using is comparable with using of statistical methods (2, 3). After this rule the result is positive, when reproducible dose-effect and/or doubling of ratio Rt/Rc is reached.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
Spontaneous reversions, negative controls (solvent), and positive controls were compared with historical controls in our laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
As the test substance is almost insoluble in any solvent, a suspension was prepared in maximum concentration given in guidelines (5000 µg per plate). This concentration was further diluted and the concentration row arised (10-5000 µg per plate) was tested for toxicity in strain TA 100 without metabolic activation. The test substance increased number of revertants gradually – none of them was toxic according to rules given in SOP M/12/10. Bacterial background was hardly evaluable because of particles of the test substance but it seemed normal.
Due to the decrease of number of revertants the first mutagenicity test in TA 100 without metabolic activation was done with higher number of doses than usually. Slow decrease was confirmed and the dose of 1500 µg per plate was toxic. The highest non- toxic dose (but dose with decreased number of revertants) -500 µg per plate- was used as maximum for the first mutagenicity experiments. This dose was diluted according to the rules given in guidelines for origination of concentration row.
No dificulties in evaluation showed at any dose, therefore the second mutagenicity experiments were done with the same doses.
All doses were dosed in amount of 0.1mL. Fresh suspensions of test substance were prepared before each experiment. The suspensions were shaken at dilution, before dosing to top agar and before pouring onto plates.

Applicant's summary and conclusion

Conclusions:
Under the above-described experimental design, the test substance Semi Dry Absorption (SDA) Product was non-mutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without as well as with metabolic activation.
Executive summary:

Test substance Semi Dry Absorption (SDA) Product was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in water for injection and assayed in doses of 5-500 ug which were applied to plates in volume of 0.1 mL.

Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance Semi Dry Absorption (SDA) Product was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.