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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
The test substance was tested in a plate incorporation and pre-incubation assay in Salmonella thypimurium strains TA100, TA102, TA97a, TA 98 and TA 1535 in presence and absence of metabolic activation. No mutagenic effects were seen in three experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants. Therefore the test substance is considered not mutagenic under the test conditions.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21-01-2014 to 06-02-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study according to the guideline under GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: LT2, TA 1535, TA 97a, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
TA97a: hisD6610, uvrB, pKM 101, rfa
TA 98: hisD3052, uvrB, pKM 101, rfa
TA 100: hisG46, uvrB, pKM 101, rfa
TA102: hisG428, pKM 101, rfa
TA1535 : hisG46, uvrB, rfa.
Metabolic activation:
with and without
Metabolic activation system:
S9 produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight ip (rTinova Biochem. Gießen (Batch no. 3067)
Test concentrations with justification for top dose:
exp 1: 0.050, 0.150, 0.500, 1.500 and 5.000 mg/plate.
exp 2 (TA 100 and TA 102): 0.016, 0.032, 0.125, 0.250 and 0.500 mg/plate
exp 3 (TA97a, TA 98 and TA 1535): 0.016, 0.032, 0.125, 0.250 and 0.500 mg/plate
Vehicle / solvent:
- Vehicle used: ethanol
- Justification for choice of solvent/vehicle: the test substance was sufficiently soluble, and no effects on the viability of the bacteria or the number of spontaneous revertants are expected.
Untreated negative controls:
yes
Remarks:
DMSO (positive control solvent)
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
yes
Remarks:
H2O
Positive controls:
yes
Remarks:
without metabolic activarion TA 97a, TA98 and TA102 : 4-Nitro-1,2-phenylene diamine; TA 1535 and TA 100 : Sodium azide ; with metabolic activation TA 97a, TA 100, TA 102 and TA 1535 : Aminoanthracene ; TA98: Benzo-a-pyrene
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, aminoanthracene
Details on test system and experimental conditions:
Cell cultures: Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem

METHOD OF APPLICATION: in medium; exp 1 preincubation; exp 2 and 3 in agar (plate incorporation)

DURATION
- Preincubation period (exp 1): 20 min
- Exposure duration: 48 h at 37 °C

SELECTION AGENT (mutation assays): histidine

CELL COUNT: visually

NUMBER OF REPLICATIONS: 4 per strain and concentration


NUMBER OF CELLS: titre determined to be 1.5E09 to 3.5E09 cells/mL
Evaluation criteria:
positive when: significant, reproducible increase of revertant colonies per plate (increase factor > 2) in at least one strain. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity .
biological relevance is taken into account first
Statistics:
none performed
Species / strain:
S. typhimurium, other: LT2 TA 1535, TA 97a, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1500 and 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test substance did not induce mutations in 5 strains of Salmonella typhimurium both in presence and absence of metabolic activation
Executive summary:

The test substance was tested in a plate incorporation and pre-incubation assay in Salmonella thypimurium strains TA100, TA102, TA97a, TA 98 and TA 1535 in presence and absence of metabolic activation. No mutagenic effects were seen in three experiments. The number of revertant colonies was not increased in comparison with the spontaneous revertants. Therefore the test substance is considered not mutagenic under the test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Ames tests on the structural analogues DNNSA and BaDNNSA were negative with and without metabolic activation. The Ames test on ZnDNNSA is one of the bridging studies used to substantiate the read-across. In view of structural similarities and the outcome of the bridging Ames test, the results of the tests on BaDNNSA are considered representative for ZnDNNSA. A chromosome aberration test and a Mouse Lymphoma cell mutation assay on BaDNNSA were both negative. Therefore it is concluded that also ZnDNNSA is not expected to exhibit mutagenic or clastogenic properties.


Justification for selection of genetic toxicity endpoint
Guideline study under GLP with the test substance

Justification for classification or non-classification

The test material, ZnDNNSA, was negative in the Ames test and a structural analogue was negative in the two other in vitro genetic toxicity tests (mouse lymphoma cell mutation assay and chromosome aberration test in human lymphocytes). Therefore ZnDNNSA is not classified for genetic toxicity according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC).