Zinc bis(benzenesulphinate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From July 21 to August 25, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(benzenesulphinate)

EC Number:

246-148-1

EC Name:

Zinc bis(benzenesulphinate)

Cas Number:

24308-84-7

Molecular formula:

C12H10O4S2Zn

IUPAC Name:

zinc bis(benzenesulphinate)

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Toxicity testing in strain TA98: 10, 100, 500, 1000, 2500, 5000 µg/plate
First mutagenicity assay: 50, 150, 500, 1500, 5000 µg/plate
Second mutagenicity assay: 15, 50, 150, 500, 1500 µg/plate

Vehicle / solvent:

- Vehicle/solvent tried to dissolve test substance: DMSO, acetonitrile, ethanol and water for injection.
- Justification for choice of solvent/vehicle: test substance was not soluble in any of them in the maximum recommended concentration 5000 μg per 0.1 ml. Test substance produced suspension in all the solvents; the best suspension (slight cloud, slow sedimentation) was observed in DMSO, thus it was used as solvent for toxicity experiment.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

no solvent

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylendiamine (TA98), N-methyl-N-nitro-N-nitrosoguanidine (E.coli)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

no solvent

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA1535, TA1537, E.coli), 2-aminofluorene (TA100 and TA98)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Preparation and using of S9
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, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/ml, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 ml/g wet liver) homogenised in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 °C. 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 µl S9 (the concentration of S9 in the S9-mix was 5.7 or 19 %). In experiments without metabolic activation only buffer was added to the top agar.

Plate incorporation test
Test procedure
100 µl of test substance of required concentration, 100 µl of 16-18 h culture of tester strain of density 10^8 -10^9 CFU/ml, 0.5 ml relevant buffer and 30 or 100 µl of S9 post-mitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3 °C. After shaking the mixture was poured into a minimal glucose agar plate.
Petri dishes were incubated of 72 h at 37±1 °C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.
For an adequate estimate of variation, triplicate plating was used at each dose level. The toxicity test, which serves for finding of optimal concentrations the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration.

Selection of doses/toxicity
Test substance was tried to dissolve in DMSO, acetonitrile, ethanol and water for injection. Test substance was not soluble in any of them in the maximum recommended concentration 5000 μg per 0.1 ml. Test substance produced suspension in all the solvents; the best suspension (slight cloud, slow sedimentation) was observed in DMSO, what was then used as solvent for toxicity experiment. For the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 98 without metabolic activation.
In application forms, turbidity occurred from concentration of 500 μg per 0.1 ml. Cloud in top agar was observable from 1500 μg per 0.1 ml. No toxicity was observed in any dose. The concentration of 5000 µg/0.1 ml was then used as maximum in the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.
At the highest dose, higher parts of precipitate were observed after adding to top agar. This dose was omitted in the second experiments and replaced by a dose of 15 μg per plate. At the same time, experiments with metabolic activation were performed with adding of 50 μl of S9 instead 30 μl.
Fresh solutions of test substance were prepared before each experiment. Concentrations of test substance solution were dosed in the volume of 0.1 ml per plate.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD guideline 471, biological relevance of results is the criterion for interpretation of results; a statistical evaluation of results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium, other: TA 1535, TA100, TA98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cytotoxicity
In preliminary cytotoxicity test, turbidity occurred in application forms from concentration of 500 μg per 0.1 ml. Cloud in top agar was observable from 1500 μg per 0.1 ml. No toxicity was observed in any dose.
In mutagenicity experiments, decrease of number of revertants was observed in the first experiment in S. typhimurium TA 1537 in the dose of 5000 µg per plate with as well as without metabolic activation. No other changes typical for toxicity as diminution of bacterial background or changes in size and distribution colonies on plates were observed and no changes were observed in the other bacterial strains in any experiments.

Applicant's summary and conclusion

Conclusions:

Non mutagenic.

Executive summary:

Method

Mutagenic potential of test substance was assessed according to OECD guideline 471.

Four Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one E. coli WP2 uvrA strain were used.

Test substance was diluted in DMSO and assayed in doses of 15 - 5000 µg per plate, which were applied to plates in volume of 0.1 ml.

The first mutagenicity experiments were performed without and with metabolic activation using a supernatant of rat liver (30 μl for S. typhimurium strains or 100 μl for E. coli per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50-5000 µg per plate.

The second mutagenicity experiments were performed with dose range of 15-1500 µg per plate and volume of S9 in experiments with metabolic activation was increased from 30 to 50 μl per plate with S. typhimurium strains and was 100 µl for E. coli.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations.

Results

Average revertant colony counts for vehicle controls were within the current historical control range for the laboratory.

Test substance was non-mutagenic for all strains without as well as with metabolic activation.