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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-isopropylidenediphenyl dicyanate
EC Number:
214-590-4
EC Name:
4,4'-isopropylidenediphenyl dicyanate
Cas Number:
1156-51-0
Molecular formula:
C17H14N2O2
IUPAC Name:
4-{2-[4-(cyanooxy)phenyl]propan-2-yl}phenyl cyanate
Test material form:
other: flaked solidified melt
Details on test material:
- Physical state: see above
- Appearance: see above
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test Substance Name: LZ 514
- Source: Lonza Ltd, Visp/Switzerland
- Lot/batch No. of test material: 5011
- Expiration date of the lot/batch: 2nd December 2017
- Manufacture date: 3rd December 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%), protected from light and humidity.
- Stability under test conditions: stable
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Supply date: 21 April 2015
Additional strain / cell type characteristics:
DNA polymerase A deficient
Cytokinesis block (if used):
n/a
Metabolic activation:
with and without
Metabolic activation system:
S9-liver fractions of rats treated with phenobarbital (PB) and β-naphthoflavone (BNF)
Test concentrations with justification for top dose:
Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and in the in the Confirmatory Mutation Test (5 strains) were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene; 4-nitro-1,2-phenylene-diamine
Details on test system and experimental conditions:
The study included a(n):
- Preliminary Compatibility Test
- Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
- Initial Mutation Test (Plate Incorporation Method)
- Confirmatory Mutation Test (Pre-Incubation Method)
Rationale for test conditions:
Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and in the in the Confirmatory Mutation Test (5 strains) were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
Evaluation criteria:
Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions was more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
Statistics:
no statistics used

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all examined strains with and without metabolic activation at several concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all examined strains with and without metabolic activation at several concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all examined strains with and without metabolic activation at several concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all examined strains with and without metabolic activation at several concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in all examined strains with and without metabolic activation at several concentrationsyyy
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitate/slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation at higher concentration

Applicant's summary and conclusion

Conclusions:
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

This study has been perfonned in accordance with the OECD Guidelines No. 471 and EC, B.13/14. The experiments were carried out with strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli (WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats. Based on the results of a solubility test, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and in the in the Confirmatory Mutation Test (5 strains) were 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate. Precipitate/slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation at higher concentrations. Inhibitory, cytotoxic effect of the test item was observed on the plates in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation at several concentrations. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.