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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

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

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

This study was conducted between 30 June 2017 and 19 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Information as provided by the Sponsor.
Identification: Lumière Violet PTM 0345N
Physical state/Appearance: Violet powder
Batch: 982432
CAS Number: 101357-19-1
EC Number: 309-916-8
Purity: Preparation containing ≥80% UVCB (treated as 100%)
Expiry Date: 01 July 2022
Storage Conditions: Room temperature in the dark
Intended use/Application: Industrial chemical
No correction for purity was required.

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fractions

Test concentrations with justification for top dose:

The test item was considered to be a UVCB* and therefore the maximum recommended dose was initially set at 5000 µg/mL. The test item was treated as 100% pure and and no purity adjustment was required in the formulations

Vehicle / solvent:

Prior to each experiment, the test item was accurately weighed, dissolved/ suspended in MEM and serial dilutions prepared

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Identity: Minimal Essential Medium (MEM) Supplier: Gibco Batch number: RNBF9655 Expiry date: 09/18

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Identity: Demecolcine (DC) CAS No.: 477-30-5 Supplier: Sigma Aldrich Batch Number: BCBR3135V Purity: >98% Expiry Date: 13 January 2022 Solvent: Sterile distilled water Concentration: 0.075 µg/mL for 24-hour continuous exposure

Details on test system and experimental conditions:

Test System and Supporting Information
Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: male, aged 27 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Batch No. PB/NF S9 30/06/17 was used in this study. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a Certificate of S9 Efficacy is presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Experimental Design and Study Conduct

Test Item Preparation
The test item was considered to be a UVCB* and therefore the maximum recommended dose was initially set at 5000 µg/mL. The test item was treated as 100% pure and and no purity adjustment was required in the formulations.
The test item was insoluble in dimethyl sulphoxide and acetone at 250 mg/mL and MEM at 50 mg/mL. The test item formed a partial solution/suspension in MEM at 25 mg/ml suitable for dosing. Prior to each experiment, the test item was accurately weighed, dissolved/ suspended in MEM and serial dilutions prepared.
Precipitate was observed when the test item formulations were dosed into media during the solubility test at and above 9.77 µg/mL. Therefore, when the preliminary toxicity test was performed the maximum dose level used was reduced to 80 µg/mL in an attempt to achieve some non-precipitating dose levels.
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 (Scott et al., 1991).
The pH and osmolality readings are presented in the following table:

Dose Concentration (µg/mL) 0 9.77 19.53 39.06 78.13 156.25 312.5 625 1250 2500
pH 7.19 7.25 7.26 7.28 7.25 7.27 7.26 7.27 7.25 7.26
Osmolality mOsm 277 275 - 283 - 277 - 275 - 272
- = Not performed for this dose concentration

The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0.313 to 80 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used for all three exposure groups was 0.078, 0.156, 0.313, 0.625, 1.25 and 2.5 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Assessments
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

Coding
The slides were coded before analysis using a computerized random number generator.

Cytokinesis Block Proliferation Index (CBPI)
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.

Scoring of Micronuclei
The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data. Experiments with human lymphocytes have established a range of micronucleus frequencies acceptable for control cultures in normal volunteer donors.
The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

Evaluation criteria:

Acceptability Criteria
The following criteria were used to determine a valid assay:
• The concurrent negative control was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9 mix.
• Cell proliferation criteria in the solvent control were considered to be acceptable.
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
• The required number of cells and concentrations was analyzed.

Statistics:

Statistical Analysis
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 0.313, 0.625, 1.25, 2.5, 5, 10, 20, 40 and 80 µg/mL. The maximum dose was selected based on the precipitating dose levels seen in the solubility test with the aim of achieving some non-precipitating dose levels.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 1.25 µg/mL, in the absence of S9 and at and above 0.625 µg/mL in the presence of S9. Precipitate was also noted on the slides at and 10 µg/mL in all three exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present up to 10 µg/mL in the 4-hour exposure in the absence of metabolic activation (S9), and up to 5 µg/mL in the 4-hour exposure in the presence of S9 and in the 24-hour exposure group. However, since precipitate was observed at lower concentrations this was the overriding factor in choosing dose levels for CBPI evaluation. The CBPI data are presented in Table 1. The test item induced no marked evidence of toxicity, up to and including the lowest precipitating dose level in any of the three exposure groups.
The selection of the maximum dose level for the Main Experiment was based the lowest precipitating dose level and was 2.5 µg/mL for all three exposure groups.

Remarks on result:

other: non-clastogenic and non-aneugenic

 

The qualitative assessment of the slides determined that there was no marked toxicity observed in the dose range selected for the main experiment and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 2.5 µg/mL in all three exposure groups.

A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 0.625 µg/mL, in all three exposure groups. This was therefore considered to be the lowest precipitating dose level and the maximum dose level selected for scoring.

 

The CBPI data for the short exposure groups and for the 24-hour exposure group confirm the qualitative observations in that no marked dose-related inhibition of CBPI was observed in any of the three exposure groups up to and including the lowest precipitating dose level. The maximum dose level selected for analysis of binucleate cells was the lowest precipitating dose level (0.625 µg/mL).

 

The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the presence of S9, the ‘A’ replicate for the CP positive control was lost due to a technical error. To compensate for this loss, an extra 1000 binucleate cells were scored from the slides of the ‘B’ replicate to give a total of 2000 binucleate cells scored for this dose level. This is considered to be acceptable under the OECD 487 guideline.

The test item did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

Conclusions:

The test item, Lumière Violet PTM 0345N, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

This report describes the results of anin vitrostudy for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes. 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4‑hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were as follows:

Exposure Group	Final concentration of test itemLumièreViolet PTM 0345N(µg/mL)
4-hour without S9	0, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5
4-hour with S9 (2%)	0, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5
24-hour without S9	0, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5

 Results

All vehicle (Minimal Essential Medium) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the lowest precipitating dose level.

Conclusion

The test item,LumièreViolet PTM 0345Nwas considered to be non-clastogenic and non‑aneugenic to human lymphocytesin vitro.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2017 - 11 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Physical state/Appearance: Brown powder
Batch: 15101887
Purity: Preparation containing ≥90% UVCB (treated as 100%)

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: University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Top agar using 0.6% Bacto agar (lot number 6147883 03/21) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

s9 fraction

Test concentrations with justification for top dose:

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. Eight test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; preincubation
- Cell density at seeding (if applicable): 0.1 mL of one of the bacterial strain cultures

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicate
NUMBER OF CELLS EVALUATED: After incubation The plates were viewed microscopically for evidence of thinning (toxicity).

Rationale for test conditions:

As the result of the preliminary experiment was deemed negative, the pre-incubation method was used in the presence and absence of metabolic activation.

Evaluation criteria:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Dunnetts Regression Analysis (* = p < 0.05)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Test item induced toxicity as a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains at and above 500 μg/plate in both the absence and presence of S9-mix. Consequently, for the second mutation test, the toxic limit of the test item was employed as the maximum dose concentration. In the second mutation test (pre-incubation method), the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 150 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were noted to all of the tester strains from 150 μg/plate (TA100 and TA1535) and 500 μg/plate (TA98, TA1537 and WP2uvrA). .
- There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Conclusions:

Lumière Violet PM 0348N was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

PV3: is not mutagenic, and is non-clastogenic and non-aneugenic based on the studies performed.