Benzenamine, 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]-, N-Me derivatives, molybdatephosphates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 27 July 2017 and 01 November 2017

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

29 July 20016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]-, N-Me derivatives, molybdatephosphates

EC Number:

268-006-8

EC Name:

Benzenamine, 4-[(4-aminophenyl)(4-imino-2,5-cyclohexadien-1-ylidene)methyl]-, N-Me derivatives, molybdatephosphates

Cas Number:

67989-22-4

Molecular formula:

C24H27N3

IUPAC Name:

4-{[4-(dimethylamino)phenyl][4-(methylamino)phenyl]methyl}-N,N-dimethylcyclohexa-2,5-dien-1-iminium; 4-{bis[4-(dimethylamino)phenyl]methylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium; 4-{bis[4-(methylamino)phenyl]methyl}-N,N-dimethylcyclohexa-2,5-dien-1-iminium

Test material form:

solid: nanoform, no surface treatment

Details on test material:

Product Number 0348N
Batch Number 15101887

Details on Nanoform
Shape
Shape Category: spheroidal
Shape: spherical
Pure Shape: Yes
Typical Composition: ≤100%
range: >0; ≤100%

Particle size distribution & range
Shape Category: spheroidal
Percentile D10, typical value: 30nm
Percentile D10, range: ≥10; ≤50nm
Percentile D50, typical value: 45nm
Percentile D50, range: ≥30; ≤100nm
Percentile D90, typical value: 60nm
Percentile D90, range: ≥45; ≤150nm
Fraction in size range 1-100nm:≥50; ≤100%

Crystallinity
structure: Amorphous
Pure structure: Yes

Specific Surface Area
Typical specific surface area: ca. 40m2/g
Range: ≥10; ≤200m2/g
Skeletal density: 1.8 g/cm3

Surface Functionalisation/treatment
surface treatment applied no

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Lumière Violet PM 0348N
Physical State / Appearance: Brown Powder
Batch: 151010887
CAS Number: 67989-22-4
EC Number: 268-006-8
Purity: Preparation containing ≥90% UVCB (treated as 100%)
Expiry Date: 28 April 2022
Storage Conditions: Room temperature, in the dark

Method

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to dilutions being prepared. The test item was supplied as a UVCB compound and tested as 100% pure. The maximum recommended dose level was 5000 µg/mL however the maximum achievable dose level was 2500 µg/mL due to formulation issues at the MRD.

Vehicle / solvent:

The vehicle control used in the main (absence of S9 mix) test was as follows:
Identity: DMSO
Supplier: Fisher
Expiry Date: 03 October 2022
Batch number 1684307

The vehicle control used in the main(presence of S9 mix) test was as follows:
Identity: DMSO
Supplier: Sigma Aldrich
Expiry Date: 01 April 2020
Batch number STBG9935

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Test System and Supporting Information
Cell Line
The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).

The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Cell Culture
Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 C with 5% CO2 in humidified air.

Cell Cleansing
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 µg/mL, 100 µM). Aminopterin (0.0178 µg/mL, 0.4 µM) and Thymidine (3.85 µg/mL, 16 µM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.

Microsomal Enzyme Fraction
Lot Number 20.08.17 was used in this study, and was pre-prepared in house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G­6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the

Preliminary Toxicity Test and the Mutagenicity Test.

Experimental Design and Study Conduct
Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and formulated in DMSO prior to dilutions being prepared. The test item was supplied as a UVCB compound and tested as 100% pure. The maximum recommended dose level was 5000 µg/mL however the maximum achievable dose level was 2500 µg/mL due to formulation issues at the MRD.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991). The pH and osmolality readings are in the following table:

Concentration µg/mL 0 9.77 19.53 39.06 78.13 156.25 312.5 625 1250 2500
pH 7.38 7.39 7.43 7.45 7.44 7.40 7.35 7.43 7.43 7.37
Osmolality mOsm 436 451 - 444 442 451 457 454 444 418

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test Procedure
Preliminary Cytotoxicity Test
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. The preliminary cytotoxicity test was performed on cell cultures plated out at 1 x 107 cells/225 cm2 flask approximately 24 hours before dosing. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask, counted at the time of dosing. On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). One flask per concentration was treated for 4-hours without metabolic activation and for 4-hours with metabolic activation (2% S9). Due to the high levels of test item precipitate observed in the solubility test the dose range of test item was set at 3.91 to 1000 µg/mL for both of the exposure groups. However, due to the excessive toxicity observed, the test was repeated using a lower dose range of 0.06 to 16 µg/mL
Exposure was for 4 hours at approximately 37 °C with a humidified atmosphere of 5% CO2 in air, after which the cultures were washed twice with phosphate buffered saline (PBS) before being detached from the flasks using trypsin. Cells from each flask were suspended in MEM with 10% FBS, a sample was removed from each concentration group and counted using a Coulter counter. For each culture, 200 cells were plated out into three 25 cm2 flasks with 5 mL of MEM with 10% FBS and incubated for 6 to 7 days at approximately 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air. The cells were then fixed and stained and total numbers of colonies in each flask counted to give cloning efficiencies (CE).
Results from the preliminary cytotoxicity test were used to select the test item concentrations for the mutagenicity experiment.

Mutagenicity Test – Main Experiment
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 107 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 106 available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
On dosing, the growth media was removed and replaced with serum-free Minimal Essential Medium (MEM). The concentrations range of test item used was 0.13 to 8 µg/mL in the absence of metabolic activation and 0.02 to 1 µg/mL in the presence of metabolic activation.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 106 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:

i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 6 to 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 µg/mL 6-Thioguanine (6-TG), to determine mutant frequency. The dishes were incubated for 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.

The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.

Calculations
The cloning efficiency (CE), % control, mutant plate counts, mutant frequency/106 (MF10-6) and mutant frequency/106 survival rate (MFSV) were calculated using the following formulae:

CE% = ( CE counts/200)x100
% Control = (CE% of Dose IDx/CE% of Dose ID0)x100
MF 10-6 for each dose = Total mutant plate counts/2
MFS 10-6 for each dose = (MF 10-6/Day 7 CE%)x100

Where:
Concentration ID0 = Vehicle control values
Concentration IDx = Concentration values
Small errors may occur when calculating mean cell concentrations and volumes for diluting; and in the calculation of means for cloning efficiency and mutant frequency; if these errors are ≤5% they are regarded to be within reasonable experimental error and considered not to affect the integrity of the study.

Evaluation criteria:

See Below

Statistics:

4.1 Statistical analysis
When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Test
A concentration range of 0.06 to 16 µg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was limited by test item-induced toxicity.
A precipitate of the test item was observed at 8 and 16 µg/mL in both exposure groups.
The results of the individual flask counts and their analysis are presented in Table 1. There was evidence of marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation.
The maximum concentration selected for the main mutagenicity experiment was therefore limited by the onset of item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines

Remarks on result:

other: non-mutagenic

Any other information on results incl. tables

The excessive levels of toxicity observed at 1 µg/mL in the absence of metabolic activation and 4, 6 and 8 µg/mL in the presence of metabolic activation, resulted in these concentrations not being plated for cloning efficiency and mutant frequency.

No precipitate of the test item was observed throughout.

There were marked concentration related reductions in the Day 0 cloning efficiency values the absence of metabolic activation and optimum levels of toxicity were achieved at 0.75 µg/mL. There were marked concentration related reductions in the Day 0 cloning efficiency values the presence of metabolic activation and optimum levels of toxicity were achieved at 2 µg/mL. There was no evidence of any marked reductions in the Day 7 cloning efficiencies in any of the surviving concentration levels, therefore indicating that residual toxicity had not occurred.

The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

The vehicle control values were all considered to be within an acceptable range, and the positive controls all gave marked increases in mutant frequency, indicating the test and the metabolic activation system were operating as expected.

Applicant's summary and conclusion

Conclusions:

The test item, Lumière Violet PM 0348N did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

The purpose of this study is to assess the potential mutagenicity of a test item, supplied by the Sponsor, on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

 Methods

Chinese hamster (V79) cells were treated with the test item at eight concentrations, in duplicate, together with vehicle (MEM culture media) and positive controls in both the absence and presence of metabolic activation. 

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by test item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

Exposure Group	Final concentration ofLumière Violet PM 0348N(µg/mL)
4-hour without S9	0, 0.03, 0.06, 0.12, 0.25, 0.5, 1.0, EMS 500, EMS 750
4-hour with S9 (2%)	0,0.13, 0.25, 0.5, 1, 2,, DMBA 1, DMBA 2

 Results

The vehicle (DMSO) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

Optimum levels of toxicity were achieved in both exposure groups

The test item, Lumière Violet PM 0348N did not induce any toxicologically significant or concentration-related increases in mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

 Conclusion

The test item, Lumière Violet PM 0348N was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.