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Genetic toxicity: in vitro

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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:
4 November 1994 to 1 December 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant study conducted in accordance with OECD guidance but with the limitation that the strains selected may not detect cross-linking mutagens.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Diammonium bis[carbonato-O]dihydroxyzirconate
EC Number:
269-682-7
EC Name:
Diammonium bis[carbonato-O]dihydroxyzirconate
Cas Number:
68309-95-5
Molecular formula:
C2H2O8Zr.2H4N
IUPAC Name:
diammonium bis[carbonato-O]dihydroxyzirconate
Details on test material:
Batch number 301 BT, reported as a 47% solution

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
33, 100, 333, 1000, 3333 and 10000 µg per plate.
Vehicle / solvent:
water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
ultra-pure water
True negative controls:
no
Positive controls:
yes
Remarks:
2-aminoanthracene, sodium azide, 9-aminoacridine, 2-nitrofluorene
Details on test system and experimental conditions:
Test Substance

Bacote 20 (Ammonium Zirconium Carbonate) Batch No. 301 BT, a colourless liquid, was received from Magnesium Elektron on 2 November 1994. Bacote 20 (purity 100%) was supplied as a 47% solution and was stored in the dark at ambient temperature.

The nominal concentration was taken to be 50%, and the stock solution was diluted accordingly for use in the mutation assays.


Positive Controls

Positive control substances used were 2-aminoanthracene (2-AAN) (Lot No. 33F-0816) from the Sigma Chemical Company Limited, Poole, Dorset, England; sodium azide (NaN3) (Lot No. 6529052) from BDH Limited, Poole, Dorset, England; 9-aminoacridine (9-AA) (Lot No. N333JE) and 2-nitrofluorene (2-NF) (Lot No. A3A) from IIT Research Institute, Chicago, Illinois 60616, USA.

The positive control substances, except sodium azide, were dissolved in dimethylsulphoxide ('AnalaR' grade from BDH Limited, Poole, Dorset, England). Sodium azide was dissolved in sterile, ultra-pure water.

Test Solution: Ultra-pure water was used to dissolve and dilute Bacote 20.

Vehicle Control: Ultra-pure water was used as the vehicle control.

Inducer: The polychlorinated biphenyl mixture, Aroclor 1254, was obtained from Monsanto (UK) Limited, Newport, Wales.

Agar Plates: The Vogel-Bonner Medium E agar plates with 2% glucose used in the Ames test were prepared in-house using BBL purified agar obtained from Becton Dickinson Limited, Oxford, England.

Animals: Male Fischer 344 rats were obtained from Charles River (UK) Limited, Margate, Kent. Bacteria

Five strains of Salmonella typhimurium were used:

S. typhimurium TA 1535 S. typhimurium TA 1537 S. typhimurium TA 1538
S. typhimurium TA 98
S. typhimurium TA 100
They 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.

METHODS

Preparation of the Metabolic Activation System

Animals
Male Fischer 344 rats (average weight 220 g) were injected once intraperitoneally with Aroclor 1254 (diluted in corn oil to a concentration of 200 mg.ml-l) at a dosage of 500 mg. kg'. They were allowed drinking water continuously, but food was withheld for 16 h before they were killed in an atmosphere of carbon dioxide 5 days after injection.

Preparation of 9,000 g Supernatant Fluid (S9 Mix) from Livers

Freshly killed animals were totally immersed in cold 2% Tego (an ampholytic detergent), then excess fluid was wiped off. The abdomen was opened and the liver removed, taking special care not to cut the gastro-intestinal tract. Livers from several animals were collected in a tared beaker containing ice-cold 0.15 M KC1.

The beaker was weighed and the livers transferred to the homogenisation vessel. A volume of ice-cold 0.15 M KC1 equivalent to 3 times the weight of the liver was added to the vessel and the livers chopped using long-handled scissors and homogenised by 8 strokes of a glass tube vessel while the Teflon pestle (radial clearance 0.14-0.15 mm) rotated at about 1,200 r.p.m. The homogenate was transferred to sterile polypropylene centrifuge tubes and spun at a relative centrifugal force of 9,000 g for 10 min at 0° to +2°C. The supernatant fluid was decanted leaving behind a thick pellet of (mainly) whole cells, nuclei and mitochondria.

Post-mitochondrial supernatant fluid was prepared in sufficient quantity for the experiment and stored, as 2 ml and 5 ml samples in sterile plastic tubes, immersed in liquid nitrogen (-196°C).

Preparation of S9 Mix

Ice-cold 0.05 M phosphate buffer, pH 7.4, was added to the following pre-weighed reagents to give final concentrations in S9 mix of:

NADP di-Na salt 4 mM (= 3.150 mg.ml-1) Glucose-6-phosphate di-Na salt 25 mM (= 7.605 mg.ml-1)

MgC12.6H20 8 mM (= 1.626 mg.ml-1)
KCI 33 mM (= 2.460 mg.ml-1)

This solution was immediately filter-sterilised by passage through a 0.45 µm Millipore filter and mixed with the liver 9,000 g supernatant fluid in the following proportion:

co-factor solution 9 parts
liver preparation 1 part

This combination of co-factors and liver preparation was called the S9 mix. 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-1). 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.HC1, 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 109 cells.ml-1) 100 µl 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 where upon 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 Bacote 20 was used:

33, 100, 333, 1000, 3333 and 10000 µg (solution supplied) 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 doses used for both these experiments were calculated on the basis of the solution supplied (nominal concentration 50%). This was diluted to give the following doses:

33, 100, 333, 1000, 3333 and 10000 µg per plate.

(NB the actual doses of Bacote 20 per plate were approximately twice those used in the toxicity test which were calculated on the basis of the weight of the solution supplied).

Triplicate plates were prepared for each bacterial strain and dose level in both the presence and absence of S9 mix.
Evaluation criteria:
Evaluation of Results

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

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 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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
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:
not applicable
Positive controls validity:
valid
Additional information on results:
Toxicity Test

No toxicity to the bacteria was observed. No precipitation of the test material occurred.

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 generally 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.

The results obtained in both experiments were similar.

Bacote 20 did not induce any significant mutagenic activity in any of the 5 bacterial strains used, in either activation condition. Increases in mutant colony numbers were noted in the first mutation assay with TA 98 and TA 1538. The number of mutant colonies was double the vehicle control
value with TA 1538 in the presence of S9 mix, and with TA 98 in the absence of S9 mix. However, the vehicle control values in these cases were low. No increases in mutant colony numbers were noted in the second mutation assay. The increases in the number of mutant colonies observed in the first assay cannot be considered significant.

No precipitation of the test material was observed.

There was no toxicity to any of the bacterial strains tested.

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

The results obtained in both experiments were similar. No significant mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Increases in mutant colony numbers were noted but these were a function of depressed vehicle control values and were not reproduced in the repeat assay.

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

It was concluded that Bacote 20 was not mutagenic to Salmonella typhimurium when tested in ultra-pure water up to a predetermined maximum limit of 10000 µg per plate.
Executive summary:

An Ames test in five strains of Salmonella typhimurium has been conducted in compliance with GLP according to OECD Guideline 471. Duplicate experiments were conducted in order to confirm the results of the study.

The results obtained in both experiments were similar. No significant mutagenic activity was observed in any of the 5 bacterial strains, in either activation condition. Increases in mutant colony numbers were noted but these were a function of depressed vehicle control values and were not reproduced in the repeat assay.

 

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

 

It was concluded that Bacote 20 was not mutagenic to Salmonella typhimurium when tested in ultra-pure water up to a predetermined maximum limit of 10000 µg per plate.