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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:
3 December 1993 - 17 December 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP study performed to a standardised guideline with a sufficient level of detail to assess the quality of the submitted data.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report date:
1994

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
This study was performed before using a strain capable of detecting cross-linking became standard for the bacterial reverse mutation method. This is not considered to affect the reliability of the study and the results are considered to be accurate and valid. Additionally the substance is not associated with mutagenic effects as observed in other in vitro tests.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Potassium Zirconium Carbonate
IUPAC Name:
Potassium Zirconium Carbonate
Test material form:
not specified

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: ues
- Periodically "cleansed" against high spontaneous background: yes
- Additional information on strains used:
The strain used were obtained in 1976 from Professor B N Ames, Department of Biochemistry, University of California, Berkeley, CA, USA, and stored in liquid nitrogen since that time until used.
All these strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. Three mutations in the histidine operon are involved:

his G 46 in TA 1535 and TA 100
his C 3076 in TA 1537
his D 3052 in TA 1538 and TA 98

his G 46 is a mis-sense mutation which is reverted to prototrophy by a variety of mutagens that cause base-pair substitutions.
his C 3076 contains a frameshift mutation which appears to have added a -G- -C- base-pair resulting in -GGGG- -CCCC-. This mutation is reverted by 9-aminoacridine, ICR-191 and epoxides of polycyclic hydrocarbons.
his D 3052 also contains a frameshift mutation with the sequence -CGCGCG- -GCGCGC- which is reverted with the deletion of 2 base-pairs -CG- -GC-. It is readily reverted by aromatic amines and derivatives.
All 5 strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharide coat of the bacterial cell surface. This deletion increases cell permeability to more hydrophobic substances and, furthermore, greatly decreases the pathogenicity of these organisms.
The second deletion, through uvrB, renders the organisms incapable of DNA excision repair and thus more susceptible to mutagenicity. These 2 deletions include the nitrate reductase (chi) and biotin (bio) genes also.
Differences between TA 1535 and TA 1538, on the one hand, and the corresponding TA 100 and TA 98 strains, on the other hand, are due to a plasmid the latter pair contains. A plasmid, R-Utrecht (pKM101), was originally shown to increase the sensitivity of the his G 46 mutation in S. typhimurium to methyl methanesulphonate and trimethyl phosphate. The particular R-factor in TA 100 and TA 98 (pKM101) carries resistance to ampicillin. It is not yet clear why the presence of this particular R-factor should increase the sensitivity of strains TA 1535 and TA 1538 to the mutagenicity of certain chemicals. The involvement of an error-prone repair mechanism has been postulated.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
33, 100, 333, 1000, 3333 and 10000 µg per plate.
Vehicle / solvent:
Sterile ultra-pure water was included as the vehicle control.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-aminoanthracene
Details on test system and experimental conditions:
Preparation of Bacteria
Samples of each strain were grown by culturing for 16 h at 37°C in nutrient broth (25 g Oxoid Nutrient Broth No. 2./litre). These cultures were kept for up to 2 days at +4°C to allow relevant checks to be performed but fresh cultures were used for the experiments.

Preparation of the Assay Plates
Diluted agar (0.6% Difco Bacto-agar, 0.6% NaCl) was autoclaved and, just before use, 5 ml of sterile 1.0 mM L-histidine.HCl, 1.0 mM biotin solution was added to each 100 ml of soft agar and thoroughly mixed. This molten agar, maintained in a water bath at 45°C, was dispensed in 2 ml volumes into small sterile tubes to which were added in order:
0.5 ml S9 mix or 0.05 M phosphate buffer, pH 7.4
0.1 ml bacteria (ca 2 x 10^9 cells/ml)
0.1 ml solvent or test solution
The tube contents, which were continually cooling, were mixed and then poured in minimal medium plates prepared in-house. These plates contained 25 ml of 1.5% BBL purified agar in Vogel-Bonner Medium E (Vogel et al (1956)) with 2% glucose. When the soft agar had set, the plates were inverted and incubated at 37°C for 2 days whereupon colonies were counted using a Biotran III automated counter (New Brunswick Incorporated, NJ, USA) at maximum sensitivity ie colonies of 0.1 mm or more in diameter counted. The plates were also examined for precipitates and, microscopically, for microcolony growth.

Toxicity Test
A toxicity test using strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. One plate of each of the following concentrations of the test material was used:
33, 100, 333, 1000, 3333 and 10000 µg per plate.

Mutation Tests
Two independent mutation tests were conducted using 5 bacterial strains (TA 1535, TA 1537, TA 1538, TA 98 and TA 100). The dose levels used in both of these experiments and selected on the basis of the results of the toxicity test, were 33, 100, 333, 1000, 3333 and 10000 µg per plate.
Triplicate plates were prepared for each bacterial strain and dose level in both the presence and absence of S9 mix.

Quality Control
At the times that the experiments were conducted, each strain was tested for its resistance to ampicillin (indicating the presence of pKM101) and sensitivity to ultraviolet (u.v.) light and crystal violet (indicating persistence of the uvrB and rfa mutations). In addition, the following control groups were established, triplicate plates being poured for each mean datum point.
1. Sterile ultra-pure water, 0.1 ml per plate, used as the test compound vehicle, in both the presence and the absence of S9 mix.
2. With S9 Mix:
2-Aminoanthracene (2-AAN), 2 µg per plate with TA 1535 and TA 1537 and 0.5 µg per plate with TA 1538, TA 98 and TA 100, used to demonstrate activity of the S9 mix and the mutability of the bacteria.
3. Without S9 Mix:
Sodium azide (NaN3), 1 µg per plate, with TA 1535 and TA 100; 2-nitrofluorene (2-NF), 1 µg per plate, with TA 1538 and TA 98; 9-aminoacridine (9-AA), 80 µg per plate, with TA 1537. These substances served as an aid to strain identification and to demonstrate the mutability of the bacteria.
Evaluation criteria:
A test was considered acceptable if for each strain:

i) the bacteria demonstrated their typical responses to crystal violet, ampicillin and u.v. light.
ii) at least 2 of the vehicle control plates were within the following ranges: TA 1535, 4-30; TA 1537, 1-20; TA 98, 10-60; TA 100, 60-200 and TA 1538, 5-35.
iii) on at least 2 of the positive control plates there were x 2 the mean vehicle control mutant numbers per plate, or in the case of TA 100, x 1.5 the mean vehicle control mutant numbers per plate.
If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required on at least 2 of the positive control plates.
iv) no toxicity or contamination was observed in at least 4 dose levels.
v) in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Where these criteria were met, a significant mutagenic response was recorded if there was:

i) for S. typhimurium strains TA 1535, TA 1537, TA 1538 and TA 98, at least a doubling of the mean concurrent vehicle control values at some concentration of the test substances and, for S. typhimurium strain TA 100, a 1.5-fold increase over the control value. If the mean colony count on the vehicle control plates was less than 10 then a value of 10 was assumed for assessment purposes. In such cases a minimum count of 20 was required before a significant mutagenic response was identified.
ii) a dose related response, although at high dose levels this relationship could be inverted because of, for example, (1) toxicity to the bacteria generally, (2) specific toxicity to the mutants and (3) inhibition of foreign compound metabolising enzymes where mutagens require metabolic activation by the liver.
iii) a reproducible effect in independent tests.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Toxicity Test
No toxicity to the bacteria was observed and there was no precipitation of the test material.

Quality Control
All strains of S. typhimurium were sensitive to crystal violet, whereas only the plasmid-containing strains, TA 98 and TA 100, were resistant to ampicillin. The strains were also tested for sensitivity to u.v. light emitted over a period of 10 s from a CAMAG u.v. lamp set at 254 nm. Increased sensitivity to u.v. light was demonstrated. These results are consistent with the known properties of these bacteria.

Vehicle Control Groups
The vehicle control values were within the normal ranges experienced in this laboratory and reported in the literature with these strains of S. typhimurium.

Positive Control Groups
The results obtained in the positive control groups were within the normal ranges expected for each bacterial strain and activation condition.

Test Rejection
All tests were acceptable according to the study criteria.

Test Material Results
The results obtained in both experiments were very similar.
No mutagenic activity was observed in any of the 5 bacterial strains, in any condition.
There was no toxicity to the bacteria and no precipitation of the test material occurred.
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

It was concluded that the test material was not mutagenic to Salmonella typhimurium when tested in sterile ultra-pure water up to a predetermined maximum limit.
Executive summary:

The test material was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at concentrations ranging from 33 to 10000 µg per plate according to OECD test Method 471.

The tests were conducted on agar plates in the presence and absence of an Aroclor 1254 induced rat liver preparation and co-factors (S9 mix) required for mixed-function oxidase activity.

Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix.

No mutagenic activity was observed in any of the 5 bacterial strains used, in any condition.

There was no toxicity to the bacteria and no precipitation of the test material was observed.

It was concluded that the test material was not mutagenic in Salmonella typhimurium when tested in sterile ultra-pure water up to a predetermined maximum limit.