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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:
from June 7, 2007 to October 1, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
see Note Deviation in the Section: Additional information on results
GLP compliance:
yes (incl. certificate)
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): Humic acids, sodium salts
- Molecular formula: not known - UVCB substance
- Molecular weight: not known - UVCB substance
- Substance type: technical product
- Physical state: solid
- Lot/batch No.: 9. 5. 2007/R
- Expiration date of the lot/batch: 05/2022
- Stability under test conditions: stable
- Storage condition of test material: dry conditions

Method

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:
a supernatant of rat liver and a mixture of cofactors
Test concentrations with justification for top dose:
50, 150, 500, 1500 and 5000 micrograms/plate
Vehicle / solvent:
Vehicle: water
- Justification for choice of solvent/vehicle: solubility of the substance in water

Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: sodium azide (1.5 ug per plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine (20 ug per plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: 2-aminofluorene (10 ug per plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (1.0 ug per plate -TA 1535, 2.5 ug per plate -TA 1537. 25 ug per plate E.coli)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine hydrochloride monohydrate (100 ug per plate)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
other: N-methyl-N-nitro-N-nitrosoguanidine (20 ug per plate)
Details on test system and experimental conditions:
The bacterial tester strains - histidine dependent Salmonella typhimurium TA 98, TA100, TA 1535, TA 1537 and tryptophan dependent strain Escherichia coli WP2 uvrA - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University Brno. Strains TA 1537 and TA 98 detect frame shift mutations, strains TA 100 and TA 1535 serve to the detection of base-pair substitution mutations and strain
E.coli WP2 uvrA detects cross-linking mutagens.

Plate incorporation test
Test procedure: 100 uL of test substance solution 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+/- 3ºC. 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+/- 1ºC.
For an adequate estimate of variation, triplicate plating was used at each dose level. Each experiment was repeated. In the case of no problems with toxicity, precipitation or dose-responsibility, doses usually remains the same as in the first experiment. In case of toxicity or precipitation, toxic (precipitating) doses are omitted. In case of mutagenicity such doses are chosen which allow construction of a dose-response curve.
Selection of doses/toxicity: Test substance was dissolved in water (aqua pro iniectione) in the maximum recommended dose 5000 ug/0.1mL. This concentration was further diluted and doses 10, 100, 500, 1000, 2500 a 5000 ug of test substance were applied to plates in volume 0.1 mL and tested for toxicity in TA 98 without metabolic activation.
The test substance coloured top agar red-brown from dose of 100 ug per plate. No signs of toxicity were observed in any dose. In doses 2500 and 5000 ug per plate, dark particles of the test substance were observed on plates.
The dose of 5000 ug per plate was chosen as maximum for mutagenicity testing, but because the test substance was not properly solved, this dose was applied in volume of 0.2 mL from solution of concentration 2500 ug/0.1 mL.
In the all experiments, the chosen doses were dosed in volume 0.1 mL with exception of the highest dose and solvent control (0.2 mL). Fresh solutions of test substance were prepared before each experiment.
Preparation and using of S9:
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g and induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL and a single injection of 500 mg/kg was administered to each rat 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL/g wet liver) homogenized in grinder and the product was centrifuged for 10 min at 9000 g. The supernatant (S9) was aliquoted into plastic tubes using sterile technique, frozen and stored at – 75 ºC. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer. Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 30 or 100 ul S9 (Concentration of S9 in S9mix was 5.7 or 16.6%). In experiments without metabolic activation only buffer was added to the top agar.
Controls:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent, negative controls contain 0.1 mL of water for injection. All the control revertant numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.
Genotypes of strains: Genotypes of each strain were controlled (plasmide pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).

Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, its using is comparable with using of statistical methods.
According to this rule the result is positive, when reproducible dose-effect and/or doubling of ratio Rt/Rc (ratio of number of revertants at tested dose to number of revertants in negative control) is reached.
Statistics:
Literature:
Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240
Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

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
Remarks:
TA100
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:
Note: Deviation:
The bacteria as well as a part of the S9 fraction used were accidentally (by outage) exposed to higher temperature (up to 2ºC), but not thawed – a deviation from SOP. Consequently performed control tests of phenotypes of bacteria and S9 effectivity showed no influence either on bacteria or on S9 fraction. The deviation had no influence on study results.

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 metabolic activation
negative without metabolic activation

Most of results obtained were with no substantial increase in number of revertants against solvent control (Rt/ Rc < 2) and no experiment with rising trend in number of revertants with increasing dose was observed.
Some experiments with increased Rt/Rc values (1.3 and 1.4) were observed, but these results have never been supported by result of the repeated experiment. On the other hand, some results also appeared with decreased Rt/Rc values, so these differences are probably made by statistical variability.
Executive summary:

Test substance 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 dissolved in water for injection and assayed in doses of 50-5000 mg which were applied to plates in amount of 0.1 or 0.2 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 Humic acids, sodium salts was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.