Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-05-15 to 2017-10-16
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Due to the toxicity of the test item at the highest dose tested 5000 μg/ plate, a supplementary dose (3000 μg/plate was studied for the first assay to evaluate the mutagenic potential of the test item at high doses.
Principles of method if other than guideline:
Due to the toxicity of the test item at the highest dose tested 5000 μg/ plate, a supplementary dose (3000 μg/plate was studied for the first assay to evaluate the mutagenic potential of the test item at high doses.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

1
Chemical structure
Reference substance name:
Bis and tris and tetra{N-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino]-N’-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino] methaniminium} [phthalocyanine(bis and tris and tetra)sulfonato-κ4N29,N30,N31,N32]cuprate(II)
Cas Number:
265115-84-2
Molecular formula:
C32H16-τN8Cu(SO3C17H22N3)τ
IUPAC Name:
Bis and tris and tetra{N-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino]-N’-[(2-ethylanilino) or 2,4(or 2,5 or 2,6)-dimethylanilino] methaniminium} [phthalocyanine(bis and tris and tetra)sulfonato-κ4N29,N30,N31,N32]cuprate(II)
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

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 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate provided by MOLTOX
Test concentrations with justification for top dose:

Solutions at 5000 µg/plate have bacteriostatic effect.compatible with the maximum tolerated.
The test item is tested at these doses (5 000, 3000, 1 500, 500, 150 and 50 μg/plate for the first assay and 5 000 , 1 500, 500, 150 and 50 μg/plate for the second assay.



Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
(stock solution of the test item prepared at 100mg/ml in Ethanol)
Controls
Untreated negative controls:
yes
Remarks:
vehicle used to solubilize the test item (ethanol)
Negative solvent / vehicle controls:
yes
Remarks:
solvent used to solubilize positive controls (NaCl 0.9%, acetone, DMSO)
True negative controls:
yes
Remarks:
absolute negative control containing no test item corresponding to the spontaneous reversion rate
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
9-aminoacridine
2-nitrofluorene
sodium azide
other: 2-anthramine and cis-platinum (II) Diamine dichloride
Details on test system and experimental conditions:
METHOD OF APPLICATION: under the direct incorporation of agar medium plate and the pre-incubation procedures


DURATION
- Preincubation period: 30 min at 37°C when the pre-incubation procedure is used.
- Exposure duration:48-72 h at 37°C
- Expression time (cells in growth medium): counting completed at the end of the exposure period in each plate


NUMBER OF REPLICATIONS: Triplicate, in parallel with negative and positive controls and the solvent of test material.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies or a clearing or diminution of the background lawn.




Evaluation criteria:
A positive result involved taking into account a dose-response effect in the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system.
The result is considered positive whenever the number of revertants of the test item treated plates is higher than those observed in the solvent treated plates, according to the following criteria:
- TA98 strain : 2 fold higher
- TA100 strain: 2 fold higher
- TA1535 strain : 3 fold higher
- TA1537 strain : 3 fold higher
- WP2 (pkM101) : 2 fold higher

The biological relevance of the results was also considered.
Statistics:
no statistics used

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
Observation: Thinning of the bacterial lawn in the presence of the highest dose tested (5 000 μg/plate), correlated with the toxicity measured at this dose.

Applicant's summary and conclusion

Conclusions:
Doses (5 000, 1 500, 500, 150 and 50 μg/plate) performed from solutions of the test item, provided by BIMA 83, do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA-) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n°471.
Executive summary:

Assay : Bacterial reverse mutation test using «Salmonella typhimurium his-» and «Escherichia coli» WP2(uvrA-)(pKM101) according to OECD guideline n° 471.

Solutions (GJV130617-S2 and GJV200617-S2) obtained from GJV240417-2, have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2(uvr A¯)(pKM101) strain.

This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out

For assay n° 1, various concentrations of GJV130617-S2 were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2, various concentrations of GJV200617-S2 were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” obtained in the laboratory.

These results validate the two tests.

There is no evidence of any increase in the number of revertant colonies in the presence of the various concentration of the test item (5 000, 1 500, 500, 150 et 50 μg/plate), without and with metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2(uvrA¯) (pKM 101).

Conclusion:

Doses (5 000, 1 500, 500, 150 and 50 μg/plate) prepared from solutions of the test item do not induce any mutagenic change in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA¯) (pKM 101) without, or with metabolic activation, according to the OECD Guidelines n° 471.

Categories Display