Reaction mass of copper and iron and zinc

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 18th November 2002 to 16th January 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

For E.coli, strain WP2 uvrA/pKM101 was used

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

First test (Range-finding)
The highest concentration of the test substance was 50 mg/ml in the chosen vehicle, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration recommended in the regulatory guidelines this assay follows

With and without S9-mix: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.

Second test
The top concentration chosen was again 5000 µg/plate, but only five concentrations were used.

With and without S9-mix: 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

The solubility of the test substance was assessed at 50 mg/ml in water and in dimethyl sulphoxide (DMSO). It was found to be insoluble and did not form a good suspension. Water (purified by reverse osmosis) containing 0.15% agar to aid suspension was, therefore, used as the vehicle for this study

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

as also Negative controls

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

as also Negative controls

True negative controls:

no

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

BACTERIAL STRAINS
The strains of S. typhimurium were obtained from the National Collection of Type Cultures, London, England.

The strain of E. coli was obtained from the National Collections of Industrial and Marine Bacteria, Aberdeen, Scotland.

PREPARATION OF S9 FRACTION
Species: Rat
Sex: Male
Strain: Sprague-Dawley derived
Source: Charles River UK Ltd.
Age: 7-8 weeks
Weight: <300 g

PREPARATION OF S9 MIX
The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM). All the cofactors were filter-sterilized before use.

MUTATION TEST PROCEDURE
First test (Range-finding)
Aliquots of 01 ml of the test dilution, positive control or negative control were placed in glass vessels. S9 mix (0.5 ml) or 0.1 M pH7.4 phosphate buffer (0.5 ml) was added, followed by 0.1 ml of a 10 hour bacterial culture and 2 ml of agar containing histidine (0.5 mM), biotin (0.5 mM) and tryptophan (0.5 mM). The mixture was thoroughly shaken and overlain onto previously prepared Petri dishes containing 25 ml minimal agar. Each Petri dish was individually labeled with a unique code corresponding to a sheet, identifying the contents of the dish. Three Petri dishes were used for each concentration. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at 37°C for ca 72 hours. After this period, the appearance if the background bacterial lawn was examined and revertant colonies counted using a Domino automated colony counter.

Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay.

STABILITY AND FORMULATION ANALYSIS
The stability of the test substance and the stability and homogeneity of the test substance in the solvent were not determined as part of the study. Analysis of achieved concentration was not performed as part of this study.

Evaluation criteria:

ACCEPTANCE CRITERIA
For a test to be considered valid the mean of the solvent/vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent solvent/ vehicle controls.

ANALYSIS OF DATA
The mean number and standard deviation of revertant colonies are calculated for all groups. The means for all treatment groups are compared with those obtained for the solvent/ vehicle control groups.

CRITERIA FOR ASSESSING MUTAGENIC POTENTIAL
IF exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent solvent/ vehicle controls, with some evidence of a positive dose-response relationship, it will be considered to exhibit mutagenic activity in this test system. No statistical analysis will be performed.

If exposure to a test substance does not produces a reproducible increase in revertant colony numbers, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis will be performed.

Statistics:

CRITERIA FOR ASSESSING MUTAGENIC POTENTIAL

If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989) and will usually be Dunnett’s test followed, if appropriate, by trend analysis. Biological significance should always be considered along with statistical significance. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Occasionally, these criteria may not be appropriate to the test data and in such cases the Study Director will use his/her scientific judegment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium 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

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

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

Positive controls validity:

valid

Additional information on results:

The absence of colonies on sterility check plates confirmed the absence of microbial contamination.

The total colony counts on nutrient agar plates confirmed the viability and high ecll density of the cultures of the individual organisms.

The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

FIRST TEST (Range-finding)
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration in either the presence of absence of S9 mix.

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test substance. A maximum exposure concentration of 5000 µg/plate was, therefore, selected for use in the second test.

SECOND TEST
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance an any concentration in either the presence or absence of S9 mix.

No visible thinning of the background lawn of non-revertant cells was obtained following exposure to the test substance.

Applicant's summary and conclusion

Conclusions:

It is concluded that, under the test conditions employed, the test substance showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

In this in vitro assessment of the mutagenic potential of the test substance, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to suspensions of the test substance diluted in water containing 0.15% agar. Water containing 0.15% agar was also used as a negative control.

 

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254 -treated rats (S9 mix). The first (range-finding) test was a standard plate incorporation assay; the second involved a pre-incubation stage.

Concentrations of the test substance up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test.

 

No evidence of mutation activity was seen at any concentration of the test substance in either mutation test.

 

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolizing activity of the liver preparations.

 

It is concluded that, under the test conditions employed, the test substance showed no evidence of mutagenic activity in this bacterial system.