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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
Study period:
Apr - May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GYEMSZI National Institue of Pharmacy
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
EC Number:
226-999-5
EC Name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
Cas Number:
5590-18-1
Molecular formula:
C22H6Cl8N4O2
IUPAC Name:
3,3'-(1,4-phenylenediimino)bis(4,5,6,7-tetrachloro-1H-isoindol-1-one)
Constituent 2
Chemical structure
Reference substance name:
mono/bis-methoxy derivatives
Molecular formula:
C22-24H6-12Cl6-8N4O2-4
IUPAC Name:
mono/bis-methoxy derivatives
Test material form:
solid: bulk
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- In the study report the old CAS number of the test substance is given.
- Lot/batch No.of test material: D1003111P2
- Appearance: yellow powder
- Expiry date: 30 July 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dimethyl sulfoxide (DMSO) was used as vehicle to prepare the test item suspensions. The test item suspensions were freshly prepared at the beginning of the experiments.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension

Method

Target gene:
his, trp
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (supplemented with cofactors) derived from phenobarbital (i.p.) and β-naphthoflavone (orally) induced rat liver
Test concentrations with justification for top dose:
Based on the results of the preliminary tests, the test item was suspended in DMSO in nominal concentrations of 50, 25, 10, 3.16, 1, 0.32 and 0.1 mg/mL to obtain seven dosing solutions (suspensions) for the test item doses. The maximum test concentration was 5000 µg test item/plate (with and without S9 mix).
Test Item concentrations tested: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was used as vehicle to prepare the test item suspensions. All dilutions of test item were made in the testing laboratory using DMSO vehicle.
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 (with S9 mix, 2 μg/plate, dissolved in DMSO, TA 98, TA 100, TA 1535 and TA 1537; and 50 µg/plate, dissolved in DMSO, E.coli WP2 uvrA); 4-nitro-o-phenylenediamine (without S9 mix, 4 μg/plate, dissolved in DMSO, TA 98)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: about 48 hours

The tests (Initial and Confirmatory Mutation Tests) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA 98 and TA 100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultues demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers is in the 10^9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
[A dose level is considered toxic if the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or the reduced revertant colony numbers are below the historical control data range and / or pinpoint colonies appear and / or reduced background lawn development occurs.]
Evaluation criteria:
The colony numbers on the control, positive control and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
Mutation Rate = (Mean revertants at the test item (or control) treatments / Mean revertants of vehicle control)

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and / or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA 100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
precipitation at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 1: Summary Table of the Results of the Range Finding Test

























































































































































































Concentrations [µg/plate]



Salmonella typhimurium tester strains



TA 98



TA 100



-S9



+S9



-S9



+S9



Mean values of revertants per plate and Mutation rate (MR)



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Untreated control



27.7



1.22



32.0



0.98



76.3



1.05



98.0



1.09



DMSO control



22.7



1.00



32.7



1.00



72.7



1.00



90.0



1.00



Ultrapure water control



-



-



-



-



79.3



1.00



-



-



5000



32.7



1.44



42.0



1.29



78.7



1.08



83.0



0.92



4000



35.0



1.54



40.7



1.24



75.7



1.04



78.7



0.87



2500



21.0



0.93



32.0



0.98



83.3



1.15



78.3



0.87



1250



19.7



0.87



32.3



0.99



79.0



1.09



83.0



0.92



625



23.7



1.04



39.3



1.20



79.0



1.09



85.7



0.95



125



25.7



1.13



27.3



0.84



78.7



1.08



89.7



1.00



25



28.3



1.25



34.0



1.04



80.0



1.10



81.0



0.90



5



27.7



1.22



27.0



0.83



80.3



1.11



82.0



0.91



NPD (4 µg)



191.3



8.44



-



-



-



-



-



-



SAZ (2 µg)



-



-



-



-



805.3



10.15



-



-



2AA (2 µg)



-



-



1093.3



33.47



-



-



1197.7



13.31



 


 


Table 2: Summary table of the results of the initial mutation test




































































































































































































































































































































































































































Concentrations [µg/plate]



Salmonella typhimurium tester strains



Escherichia coli


WP2 uvrA



TA 98



TA 100



TA 1535



TA 1537



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean values of revertants per plate


Mutation rate (MR)



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Untreated control



34.7



1.14



35.0



1.19



85.0



1.21



72.3



0.95



9.0



1.35



9.3



1.22



6.7



0.91



9.3



1.22



16.7



1.04



30.3



1.21



DMSO control



30.3



1.00



29.3



1.00



70.0



1.00



76.3



1.00



6.7



1.00



7.7



1.00



7.3



1.00



7.7



1.00



16.0



1.00



25.0



1.00



Ultrapure water control



-



-



-



-



78.0



1.00



-



-



6.0



1.00



-



-



-



-



-



-



19.0



1.00



-



-



5000



26.7



0.88



50.7



1.73



63.0



0.90



78.0



1.02



7.0



1.05



6.7



0.87



5.0



0.68



5.7



0.74



13.7



0.85



16.7



0.67



2500



25.7



0.85



45.0



1.53



84.7



1.21



85.0



1.11



6.7



1.00



9.3



1.22



8.0



1.09



7.3



0.96



11.7



0.73



16.0



0.64



1000



18.7



0.62



34.0



1.16



84.7



1.21



84.3



1.10



6.0



0.90



13.7



1.78



6.3



0.86



7.7



1.00



13.0



0.81



21.3



0.85



316



30.0



0.99



35.7



1.22



76.3



1.09



81.7



1.07



4.7



0.70



7.3



0.96



9.0



1.23



6.0



0.78



20.7



1.29



22.7



0.91



100



36.7



1.21



36.0



1.23



66.7



0.95



91.3



1.20



8.3



1.25



8.0



1.04



8.3



1.14



7.3



0.96



13.7



0.85



18.3



0.73



31.6



30.3



1.00



39.0



1.33



78.0



1.11



89.3



1.17



6.3



0.95



7.7



1.00



6.0



0.82



8.7



1.13



17.7



1.10



24.7



0.99



10



27.7



0.91



40.3



1.38



75.7



1.08



80.0



1.05



7.3



1.10



8.3



1.09



5.7



0.77



7.3



0.96



13.0



0.81



20.0



0.80



NPD (4 µg)



166.3



5.48



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



SAZ (2 µg)



-



-



-



-



889.3



11.40



-



-



920.0



153.33



-



-



-



-



-



-



-



-



-



-



9 AA (50 µg)



-



-



-



-



-



-



-



-



-



-



-



-



333.3



45.45



-



-



-



-



-



-



MMS (2 µL)



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



652.7



34.35



-



-



2 AA (2 µg)



-



-



910.7



31.05



-



-



1480.3



19.39



-



-



116.0



15.13



-



-



137.3



17.91



-



-



-



-



2 AA (50 µg)



-



-



-



-



 



 



-



-



-



-



-



-



-



-



-



-



-



-



288.3



11.53



 


 


Table 3: Summary table of the results of the confirmatory mutation test




































































































































































































































































































































































































































Concentrations [µg/plate]



Salmonella typhimurium tester strains



Escherichia coli


WP2 uvrA



TA 98



TA 100



TA 1535



TA 1537



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



-S9



+S9



Mean values of revertants per plate


Mutation rate (MR)



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Mean



MR



Untreated control



25.7



1.12



40.3



1.14



91.0



1.19



99.0



1.02



11.7



1.67



15.0



1.36



4.7



1.08



7.3



0.92



17.3



0.75



29.3



0.87



DMSO control



23.0



1.00



35.3



1.00



76.3



1.00



96.7



1.00



7.0



1.00



11.0



1.00



4.3



1.00



8.0



1.00



23.0



1.00



33.7



1.00



Ultrapure water control



-



-



-



-



83.7



1.00



-



-



8.7



1.00



-



-



-



-



-



-



21.7



1.00



-



-



5000



18.0



0.78



42.3



1.20



68.7



0.90



95.3



0.99



7.7



1.10



9.7



0.88



5.7



1.31



7.7



0.96



13.3



0.58



29.7



0.88



2500



12.7



0.55



35.3



1.00



66.7



0.87



98.7



1.02



6.0



0.86



9.0



0.82



6.7



1.54



5.0



0.63



21.3



0.93



29.7



0.88



1000



20.0



0.87



35.3



1.00



71.3



0.93



88.3



0.91



8.0



1.14



10.7



0.97



5.7



1.31



5.3



0.67



16.3



0.71



29.0



0.86



316



32.3



1.41



33.0



0.93



77.0



1.01



91.3



0.94



7.3



1.05



10.0



0.91



4.7



1.08



9.0



1.13



20.7



0.90



31.0



0.92



100



30.7



1.33



28.0



0.79



66.7



0.87



75.7



0.78



9.0



1.29



10.3



0.94



4.7



1.08



6.0



0.75



16.7



0.72



27.3



0.81



31.6



29.3



1.28



35.0



0.99



60.7



0.79



85.0



0.88



6.0



0.86



11.0



1.00



4.7



1.08



8.3



1.04



17.3



0.75



35.3



1.05



10



21.0



0.91



33.7



0.95



61.7



0.81



78.3



0.81



7.3



1.05



9.3



0.85



4.7



1.08



7.3



0.92



16.0



0.70



28.3



0.84



NPD (4 µg)



193.3



8.41



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



SAZ (2 µg)



-



-



-



-



1169.3



13.98



-



-



730.0



84.23



-



-



-



-



-



-



-



-



-



-



9 AA (50 µg)



-



-



-



-



-



-



-



-



-



-



-



-



525.0



121.15



-



-



-



-



-



-



MMS (2 µL)



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



645.3



29.78



-



-



2 AA (2 µg)



-



-



913.3



25.85



-



-



828.3



8.57



-



-



121.3



11.03



-



-



173.7



21.71



-



-



-



-



2 AA (50 µg)



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



-



202.0



6.00


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 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital / ß-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate.

In the performed preliminary experiments no cytotoxicity of the test item was observed. At the concentration choice the guideline criterion for non-cytotoxic substances was taken into consideration where the recommended maximum test concentration is 5000 µg/plate. The test item was not soluble, and precipitated at the concentration range of 5000 - 1000 µg/plate. The obtained precipitate interfered the scoring, therefore the necessity of an additional microscopic analysis of the plates was considered.

In the Initial and Confirmatory Mutation Tests the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

The revertant colony numbers of vehicle control plates with and without S9 Mix were within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

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 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.