Diammonio(ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 24th to November 24th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diammonio(ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium

EC Number:

220-168-0

EC Name:

Diammonio(ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium

Cas Number:

2650-18-2

Molecular formula:

C37H42N4O9S3

IUPAC Name:

diammonium 2-[(4-{ethyl[(3-sulfonatophenyl)methyl]amino}phenyl)[(1E,4E)-4-{ethyl[(3-sulfonatophenyl)methyl]iminiumyl}cyclohexa-2,5-dien-1-ylidene]methyl]benzene-1-sulfonate

Test material form:

semi-solid (amorphous): gel

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

DOSE FIRST MAIN ASSAY -PLATE INCORPORATION METHOD:
TA98, TA100, TA 1535, TA 1537, WP2 uvrA (±S9): 5000, 1500, 500, 150, 50 µg/plate
The test substance is well soluble in water, so it could be dissolved in the highest concentration 5 mg per plate (50 mg per mL) recommended in OECD TG 471. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 98 without metabolic activation. No toxicity or precipitation was observed in any dose so the highest concentration could be used as maximum for the first mutagenicity experiments. Further doses were diluted with factor approximately 2-√10.

DOSE SECOND MAIN ASSAY - PRE-INCUBATION METHOD:
TA98, TA100, TA 1535, TA 1537, WP2 uvrA (±S9): 5000, 4000, 3000, 2000, 1000 µg/plate
The dose-range of Main Assay II was slightly modified to take into account the results of Main Assay I: the same maximum dose was used but single concentrations were increased.

Vehicle / solvent:

Water to dissolve the test item and for injections

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 4-nitro-o-phenylenediamine, N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without metabolic activaton

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene; 2-aminoanthracene

Remarks:

with metabolic activaton

Details on test system and experimental conditions:

BACTERIAL STRAINS
The bacterial tester strains Salmonella typhimurium TA 1535 (CCM 3814, lot. No. 2101200916917), TA 98 (CCM 3811, lot No. 0102201220053), TA 100 (CCM 3812, lot No. 0102201220054) and TA 1537 (CCM 3815, lot No. 2101200916918) as well as Escherichia coli WP2 uvrA (CCM 4751, lot No. 2104200512732), - were obtained from Czech Collection of Microorganisms (CCM) of Masaryk University, Brno.
Strains TA 98 and TA 1537 detect frame shift mutations, strains TA 100 and TA 1535 serve to detection of base-pair substitution mutations, and strain E.coli WP2 uvrA detects cross-linking mutagens.
Genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).

MEDIA
Nutrient Broth for microbiology, Merck, Lot. VM648943433, exp. 25/0354/2019
Nutrient agar, Merck,VM664850449, exp. 05/11/2019
Agar-agar, Merck, Lot. K47292515 604, minimum shelf life 12/2019
Procedures of media preparation are described in detail in internal SOP M/12.

S9 TISSUE
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg·mL-1, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 mL.g-1 wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 °C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 mL of buffer with NADP and glucoso-6-phosphate and 30 or 100 µL S9 (the concentration of S9 in the S9mix was 5.7 or 16.7%). In experiments without metabolic activation only buffer was added to the top agar.

CONTROLS
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 mL of water for injection. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. The test substance is well soluble in water, so it could be dissolved in the highest concentration 5 mg per plate (50 mg per mL) recommended in OECD TG 471. For toxicity experiment the highest concentration was diluted to the other 5 concentrations in 3 digit places interval. The concentration row was tested for toxicity in strain TA 98 without metabolic activation.

MAIN ASSAYS
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
- FIRST MAIN ASSAY-PLATE INCORPORATION METHOD: 100 µL of the test item of required concentration, 100 µL of 16-18 h culture of tester strain of density 108-109 CFU/mL, 0.5 mL relevant buffer and 30, or 100 µL of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 mL of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3 °C. After shaking the mixture was poured into a minimal glucose agar plate.
- SECOND MAIN ASSAY- PRE-INCUBATION METHOD:
0.5 mL of relevant buffer, 100 µL of the test item of required concentration, 100 µL of 16-18 h culture of tester strain of density 108-109 CFU/mL and 30/100 µL of S9 post mitochondrial fraction were mixed and shaken at 37±1 °C for 30 minutes (preincubation). Then, 2 mL of molten top agar (with trace of histidine or tryptophan) was added and the mixture was poured into a minimal glucose agar plate.

INCUBATION AND SCORING
Petri dishes were incubated of 72 h at 37±1 °C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000. For an adequate estimate of variation.

ACCEPTANCE CRITERIA
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc (ratio of number of revertants at tested dose to number of revertants in negative control) is reached.

Evaluation criteria:

An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD TG 471, 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.

Statistics:

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.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA

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:

RESULTS OF PLATE INCORPORATION AND PRE-INCUBATION ASSAY.
No increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.
The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

CYTOTOXICITY
In preliminary cytotoxicity test, no toxicity occurred in any concentration.
In mutagenicity experiments, no decrease of number of revertants or diminution of bacterial background was observed in any bacrerial strain/dose.

ACCEPTANCE CRITERIA
All acceptance criteria were fulfilled.

EVALUATION OF THE RESULTS
The test item was non-mutagenic for all the used tester strains without as well as with metabolic activation.

Applicant's summary and conclusion

Conclusions:

The test item was non-mutagenic for all the used tester strains without as well as with metabolic activation.

Executive summary:

The test item was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coliWP2 uvrA strain were used.

The test item was non-mutagenic for all the used tester strains without as well as with metabolic activation.

CYTOTOXICITY

In preliminary cytotoxicity test, no toxicity occurred in any concentration.

In mutagenicity experiments, no decrease of number of revertants or diminution of bacterial background was observed in any bacrerial strain/dose.