Reaction product of tin, citric acid and nitric acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

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

Test material information

Constituent 1

Reference substance name:

Reaction product of tin, citric acid and nitric acid

EC Number:

944-119-4

Molecular formula:

not available

IUPAC Name:

Reaction product of tin, citric acid and nitric acid

Test material form:

liquid

Method

Target gene:

his, trp

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:

phenobarbital and ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: TA 1535, TA 100, TA 1537 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 3333 and 10000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: Due to an update on the content of the test substance, the non-toxic tester strains were repeated. The doses were adjusted to the new content and the results of the 1st Experiment.

3rd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 3333 and 10000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.

4th Experiment
Strains: TA 1537 and TA 98
Doses: 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (TA 1537 without S9 mix)
0; 10; 33; 100; 333; 1000 and 3333 mg/plate (TA 1537 and TA 98 with S9 mix)
Type of test: Preincubation test
Number of plates: 3 test plates per dose or per control
Reason: Bacteriotoxicity was observed in the preincubation test.

Vehicle / solvent:

- Vehicle used: water
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine; 9-aminoacridine; 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min (PIT)
- Exposure duration: 48 - 72 h (SPT & PIT)

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants (factor ≤ 0.6), clearing or diminution of the background lawn (= reduced his- or trp- background growth)

Evaluation criteria:

Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings in the 1st Experiment.

Toxicity:
Toxicity detected by a
- decrease in the number of revertants (factor ≤ 0.6)
- clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility:
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification / substantiation.

Statistics:

Acceptance criteria:
Generally, the experiment was considered valid if the following criteria were met
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies compatible with the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria:
The test substance was considered positive in this assay if the following criteria were met
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Species / strain:

other: Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: please refer to "Any other information on results incl. tables"

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No precipitation of the test substance was found with and without S9 mix.

Any other information on results incl. tables

A bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 2500 μg/plate onward.

In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 1000 μg/plate onward.

Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	without	-	-	-	2500 -5000	-
	with	-	-	-	2500 -5000	-
2nd-SPT	without	3333 -10000	-	-	n.t.	-
	with	3333 -10000	10000	10000	n.t.	-
3rd-PIT	without	10000	3333 -10000	1000 -10000	3333 -10000	3333 -10000
	with	1000 -10000	3333 -10000	3333 -10000	3333 -1000	1000 -10000
4th-PIT	without	n.t.	n.t.	1000	n.t.	n.t.
	with	n.t.	n.t.	3333	3333	n.t.

- = no adverse effect observed

n.t. = not tested

Reduced background growth was observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	without	5000	5000	5000	5000	5000
	with	5000	5000	5000	5000	5000
2nd-SPT	without	10000	10000	10000	n.t.	10000
	with	10000	10000	10000	n.t.	10000
3rd-PIT	without	3333 -10000	3333 -10000	3333 -10000	3333 -10000	3333 -10000
	with	3333 -10000	3333 -10000	3333 -10000	3333 -10000	3333 -10000
4th-PIT	without	n.t.	n.t.	-	n.t.	n.t.
	with	n.t.	n.t.	3333	3333	n.t.

- = no adverse effect observed

n.t. = not tested