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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1983)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
425-240-7
EC Name:
-
Molecular formula:
C28H42O8Cl2Co2N12Zn4
IUPAC Name:
Zinc hexacyanocobaltate(III), tertiary butyl alcohol/polypropylene glycol complex

Method

Target gene:
histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium, other: TA98 , TA100, TA1535, TA97a, TA102
Metabolic activation:
with and without
Metabolic activation system:
Liver S-9 fraction from Aroclor 1254 induced rat (= S9-mix)
Test concentrations with justification for top dose:
Exp. 1 (with and without metabolic activation) - for all strains: 8, 40, 200, 1000 and 5000 µg/plate
Exp. 2 (without metabolic activation) - for TA98, TA100, TA1535: 156.25, 312.5, 625, 1250, 2500 µg/plate; - for TA97a, TA102: 31.25, 62.5, 125, 250, 500 µg/plate
Exp. 2 (with metabolic activation) - for TA98, TA97a, TA102: 6.25, 12.5, 25, 50, 100 µg/plate; - for TA100, TA1535: 156.25, 312.5, 625, 1250, 2500 µg/plate
Vehicle / solvent:
sterile purified water (water purified by reverse osmosis);
As no adequate solvent could be readily identified, all test treatments were performed using a suspension.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Nitroflourene (5 µg/plate; only TA98), Na-azide (2 µg/plate; TA1535 and TA100), 2-aminoacridine (50 µg/plate; TA97a), Glutaraldehyd (25 µg/plate; TA102), 2-aminoanthracene (5 µg/plate; at least one strain).
Remarks:
The positive controls 2-nitroflourene, Na-azide, 2-aminoacridine and Glutaraldehyd were used without S9-mix, 2-aminoanthracene was used with S9-mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
Two independent experiments performed, each including a 20 min. (37°C) liquid pre-incubation step.
The test protocol was designed to maximise the detection of any mutagenic activity associated with the divalent metals known to be complexed within the test material. Modifications include pre-incubation rather than plate incorporation treatments, the use of HEPES rather than Phosphate buffer in the assay system and the inclusion of a tester strain sensitive to the mutagenic activity of these metals (For reference see Pagano and Zeiger, Environmental and Molecular Mutagenesis 19, 1992, 139-146).

DETERMINATION OF CYTOTOXICITY:
mutant count, inspection of backround lawn
Evaluation criteria:
The test article was considered to be mutagenic if: 1) the assay was valid (negative and positive controls valid and no more than 5% of the plates lost through contamination or some other unforeseen event) 2) Dunnett's test gave a significant response (p
Statistics:
The m-statistic was calculated to check that the data were Poisson-distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA97a, TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Particulate matter was observed in some of the plates, which was not unexpected as the material was applied as a suspension.

RANGE-FINDING/SCREENING STUDIES:
An initial toxicity range-finder experiment was carried out in strain TA100 only, using final concentrations of 8-5000 µg/plate, plus negative (vehicle) and positive controls. Evidence of toxicity was observed only at 5000 µg/plate, and this same dose range was used for the main testings.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Exp. 1, toxicity was seen down to 200 µg/plate in the presence of S9-mix and down to 1000 µg/plate in the absence of S9-mix, in three of the four strains tested (two strains in the case of this toxicity in the absence of S9-mix). In Exp. 2, the concentrations were reduced accordingly although toxicity was still apparent at the highest levels (and at the two highest concentrations tested in TA100 in the presence of S9-mix).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Executive summary:

The test substance was investigated in a Bacterial Reverse Mutation (Ames) Test according to OECD TG 471 on S. thyphimurium TA 98, TA100, TA1535, TA97a and TA102. No increase in revertant numbers was detected, in either the absence or presence of metabolic activation. This was the case even though the test protocol was designed to maximise the detection of any mutagenic activity associated with the divalent metals known to be complexed within the test material (pre-incubation rather than plate incorporation, use of HEPES rather than Phosphate buffer, inclusion of a tester strain sensitive to the mutagenic activity of these metals). Therefore the substance was considered to be non-mutagenic.

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