2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

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:

8 Jun 2016 to 30 Jun 2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

EC Number:

276-586-9

EC Name:

2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

Cas Number:

72319-18-7

Molecular formula:

C23H26N3.HO4S

IUPAC Name:

2-[2-[4-[(2-cyanoethyl)methylamino]phenyl]vinyl]-1,3,3-trimethyl-3H-indolium hydrogen sulphate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name of test substance as used in report: Basic Red 14 sulfate
- Test substance No.: 16/0122-1
- Batch identification: Lot 4030158820
- Content: 100 g/100 g (Titration)
- Water content: 0.09 g/100 g
- Date of production: 31 Mar 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Guaranteed until Mar 2018
- Solubility and stability of the test substance in the vehicle: The stability of the test substance in the vehicle water was not determined analytically, because the test substance was administered immediately after preparation and is usually stable.

OTHER SPECIFICS:
- Physical state, appearance: Solid, violet to sparkling dark blue

Method

Target gene:

S. typhimurium: his
E. coli: trp

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: The Salmonella strains TA 1535, TA 100, TA 1537 and the Escherichia coli strain were obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014. The Salmonella strain TA 98 was obtained from Moltox Molecular Toxicology on 07 Jan 2015.
- Suitability of cells: The use of the strains mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
- Methods for maintenance in cell culture: Deep-frozen (-70°C to -80°C) bacterial cultures were thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria cultures was determined. Fresh cultures of bacteria were grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). These cultures grown overnight were kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
- The Salmonella strains were checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity (Δ uvrB); ampicillin resistance (R factor plasmid).
- E. coli WP2 uvrA was checked for UV sensitivity.
- Histidine and tryptophan auxotrophy was checked in each experiment via the spontaneous rate.

MEDIA USED
- Type and identity of media: nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital (i.p.) and β-naphthoflavone (oral) induced rats livers

Test concentrations with justification for top dose:

JUSTIFICATION
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in EXPERIMENT 1. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

EXPERIMENT 1
- Without S9 mix: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
- With S9 mix: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

EXPERIMENT 2
- Without S9 mix: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate
- With S9 mix: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate

EXPERIMENT 3
- Without S9 mix: 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate
- With S9 mix: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate

EXPERIMENT 4 (strain TA 98 only)
- Without S9 mix:0; 3.3; 10; 33; 100; 333 and 1000 μg/plate

Vehicle / solvent:

- Vehicle used: ultrapure water
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, water was used as vehicle.

OTHER
- To achieve a clear solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly.
- All test substance formulations were prepared immediately before administration.

Controls

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION
- EXPERIMENT 1: in agar (plate incorporation)
- EXPERIMENT 2-4: preincubation

DURATION
- Preincubation period (EXPERIMENT 2-4): 20 minutes at 37°C
- Exposure duration (EXPERIMENT 1-4): 48 – 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS
- 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
Following incubation, the bacterial colonies (his+ revertants for Salmonella typhimurium, trp+ revertants for E. coli) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

EVALUATION
- Mutagenicity: Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.
- Toxicity: Toxicity detected by a decrease in the number of revertants (factor ≤ 0.6) and/or clearing or diminution of the background lawn (= reduced his- or trp- background growth) was recorded for all test groups both with and without S9 mix in all experiments. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.
- Solubility: If precipitation of the test material was observed, it would be recorded.

Rationale for test conditions:

EXPERIMENT 2
No mutagenicity was observed in the standard plate test.

EXPERIMENT 3
Due to technical reason, an evaluation for the most part of EXPERIMENT 2 was not possible. Furthermore, bacteriotoxicity was observed in the evaluable tester strain, therefore the whole experiment was repeated with adjusted doses. The results of EXPERIMENT 2 are not reported.

EXPERIMENT 4
Inconclusive values were observed in the preincubation test.

Evaluation criteria:

ACCEPTANCE CRITERIA
Generally, the experiment was considered valid if the following criteria were met:
- The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
- The sterility controls revealed no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- Fresh bacterial culture containing approximately 10^9 cells per mL were used.

ASSESSMENT CRITERIA
The test substance was considered positive in this assay if the following criteria were met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the range of the historical negative control data under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 1000 μg/plate onward, see 'Any other information on results inc. tables'

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500-5000 μg/plate, see 'Any other information on results inc. tables'

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

TA 98: mutagenicity observed in EXPERIMENT 3, however not reproducible in EXPERIMENT 4, therefore biological irrelevant. See also Ádditional information on results'.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 333 μg/plate onward, see 'Any other information on results inc. tables'

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000-2500 μg/plate, see 'Any other information on results inc. tables'

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found with and without S9 mix.

ADDITIONAL INFORMATION ON MUTAGENICITY
Using the tester strain TA 98 without S9 mix in the preincubation test (EXPERIMENT 3) increased numbers of his+ revertants were observed at concentrations of 33 and 100 μg/plate (factors 3.6 and 2.5, respectively). In a repeat experiment (EXPERIMENT 4) this finding was not reproduced and therefore these findings have to be regarded as biological irrelevant.

Remarks on result:

other: Standard plate test (EXPERIMENT 1)

Any other information on results incl. tables

Table 1. Decreased revertant numbers were observed at following concentrations (µg/plate)

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1	Without	1000-5000	5000	5000	5000	2500-5000
1	With	5000	5000	2500-5000	2500-5000	2500-5000
3	Without	1000	-	333-1000	-	-
3	With	1000-2500	-	333-2500	-	1000-2500
4	Without	n.t.	n.t.	n.t.	1000	n.t.
4	With	n.t.	n.t.	n.t.	n.t.	n.t.

- = no adverse effect observed

n.t. = not tested

Table 2. Reduced background growth was observed at following concentrations (µg/plate)

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1	Without	5000	5000	5000	5000	-
1	With	5000	5000	5000	5000	-
3	Without	1000	1000	1000	1000	1000
3	With	2500	2500	2500	2500	2500
4	Without	n.t.	n.t.	n.t.	-	n.t.
4	With	n.t.	n.t.	n.t.	n.t.	n.t.

- = no adverse effect observed

n.t. = not tested