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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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

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

Applicant's summary and conclusion