Octasodium 2,2'-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(1-hydroxy-3,6-disulphonatonaphthalene-2,8-diyl)azo]]bisnaphthalene-1,5-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bayscript Magenta BB is not mutagenic in vitro 1) in a bacterial test and 2) in a HPRT test with V79 cells and was considered to be non-clastogenic and non-aneugenic to human lymphocytes.

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:

Experimental start date: 09 November 2017. Experimental completion date: 06 December 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian forward mutation assay

Specific details on test material used for the study:

Chemical Name: 1,5-Naphthalenedisulfonic acid, 2,2'-[1,4-phenylenebis[imino (6-chloro-1,3,5-triazine-4,2-diyl)imino(8-hydroxy -3,6-disulfo-1,7-naphthalenediyl)-2,1-diazenediyl]]bis-,sodium salt (1:8)
Appearance: Solid brown to black, golden shiny
Expiry / Retest Date: 17 June 2018
Storage Conditions: At room temperature

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. Especially 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%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Cell Cultures
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni¬cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before free¬zing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. About 2-3 x10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

Culture Medium
For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Test on Toxicity
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 20.2 µg/mL and 2581.0 µg/mL were used. The highest concentration was chosen with respect to the current OECD guideline 476 regarding the purity of the test substance (77.5%).
Cytotoxic effects indicated by a relative cloning efficiency below 50% were observed at 645.3 µg/mL and above without metabolic activation, and at 1290.5 µg/mL and above after 4 hours treatment.

Main Test
without S9 mix: 162.5, 325.0 487.5, 650.0, 975.0* μg/mL
with S9 mix: 78.1, 156.3, 312.5, 625.0, 937.5 μg/mL
Based on the cytotoxicity observed in the pre-experiment, 1300 µg/mL without metabolic activation and 1875.0 µg/mL with metabolic activation were chosen as top concentrations for the main experiment. The individual concentrations were generally spaced by a factor of 2. Narrower spacing was used at higher concentrations to cover the range of cytotoxicity more closely.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations

Vehicle / solvent:

The test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Remarks:

300 µg/mL = 2.4 mM

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolica activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Remarks:

2.3 µg/mL = 8.9 µM

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Pre-Test on Toxicity
A pre-test was performed in order to determine the toxicity of the test item. In addition the pH and osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In this pre-test approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

Seeding
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2% in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2 x 10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

Treatment
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0 x 10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment as described below.
Three or four days after first sub-cultivation approximately 2.0 x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 6 days five 75 cm² cell culture flasks were seeded with about 4 to 5 x 10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and therefore the test chemical response will be concluded to be equivocal.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was not performed since none of the mutation frequencies calculated for the evaluated test item concentrations exceeded the 95% confidence interval.
However, both, biological and statistical significance were considered together

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item Bayscript Magenta BB was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.
Cytotoxic effects indicated by a relative total growth of less than 50% of relative survival as mean of both parallel cultures occurred at 650.0 µg/mL and above without metabolic activation, and at 625.0 µg/mL and above with metabolic activation. The data at 975.0 µg/mL without metabolic activation are rejected as the relative adjusted cloning efficiency I was below 10%.
In the main experiment with and without S9 mix the mean mutant frequency of the solvent control was 19.9 and 21.6 mutants per 10^6 cells. The values were well within the 95% confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the groups treated with the test item ranged from 9.8 up to 23.9 mutants per 106 cells.
No relevant increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration.
The 95% confidence interval was not exceeded at any experimental point. All mutant frequencies remained well within the range of the historical solvent control data.
The linear regression analysis showed no significant dose dependent trend of the mutation frequency.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

The main experiment was analyzed at the following concentrations:

S9 mix	concentrations in µg/mL
	Main experiment / exposure period 4 hours
-	162.5	325.0	487.5	650.0	975.0*
+	78.1	156.3	312.5	625.0	937.5

*Not taken into evaluation due to strong toxic effects (rel. adjusted CE I <10%)

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Bayscript Magenta BB is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Bayscript Magenta BB to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (2581 µg/mL) was chosen with respect to the OECD guideline 476 (2016) regarding the purity of the test item. The concentration range of the main experiment was limited by cytotoxicity observed in the pre-experiment.

No relevant increase in mutant colony numbers/10⁶cells was observed in the main experiment up to the maximum concentration.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Bayscript Magenta BB is considered to be non-mutagenic in this HPRT assay.

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Bayscript Magenta VPSP 25032
Purity: more than 99%
Powder
Lot WDP9613 Pure Powder
Stable in water

Target gene:

His

Species / strain / cell type:

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Up to the limit concentration of 5000 µg/plate

Vehicle / solvent:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Statistics:

Dunnett's one-side test

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 100

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

E. coli WP2 uvr A

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

Executive summary:

The mutagenic activity of BAYSCRIPT MAGENTA VPSP 25032 was examined in the reverse mutation test by using bacterial strains that have different two patterns of the mutation. One is the base-pair substitution of Salmonella typhimurium TA100, TA1535 and Escherichia coli WP2 uvrA. The other is the frameshift mutation of Salmonella typhimurium TA98 and TA1537. The test was conducted in direct plate incorporation methods in all bacterial strains in both the presence of metabolic activation and the absence of metabolic activation. In this test, two statistical analyses that are Dunnett’s multiple comparison method (one-side test) and linear regression method were prepared to evaluate the test results. The number of the revertant colonies of each bacterial strains of each dose was compared with the negative control, and statistically significant in the number of the revertant colonies between those two groups was analyzed first by the multiple comparison method. When the statistical significant difference was obtained by the multiple comparison method, the specific dose-relativity was analyzed by linear regression method. The test substance was judged as positive for mutagenic activity when biologically significant increases such as significant increase in the number of the revertant colonies with clear dose-relativity and reproducibility was obtained. The results of the test are as follows:

1. Biologically significant increase in the number of the revertant colonies compared with the negative control was not detected in any bacterial strains regardless of the presence of metabolic activation or the absence of metabolic activation.

2. The values of the negative controls and positive controls were appropriate value in comparison with historical data of our laboratory. Furthermore, all of the positive controls such as 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminocaridine, benzo[α]pyrene and 2-aminoanthracene increased the number of the revertant colonies sufficiently compared with the negative control with all bacterial strains, respectively. These results indicate that the test has been properly carried out.

3. From the foregoing results, it is concluded that the mutagenic activity of BAYSCRIPT MAGENTA VPSP 25032 is judged to be negative under the test conditions employed.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 05 June 2017 Experimental completion date: 10 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)

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:

Identification: Bayscript Magenta BB
1,5-Naphthalenedisulfonic acid, 2,2’-[1,4-phenylenebis[imino(6-chloro-1,3,5-triazine-4,2-diyl)imino(8-hydroxy-3,6-disulfo-1,7-naphthalenediyl)-2,1-diazenediyl]]bis-, sodium salt (1:8)
Physical state/Appearance: Metallic bronze coloured solid flakes
Expiry Date: 17 June 2018
Formulated concentrations were adjusted to allow for the stated water/ organic impurities content of the test item (Currenta study No. 2016/0063/01).

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: whole blood

Details on mammalian cell type (if applicable):

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: female, aged 26 years
Main Experiment: female, aged 30 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).

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

The molecular weight of the test item was given as 1774.2 g/mol, therefore, the maximum dose level was 2000 μg/mL, the maximum recommended dose level.

Preliminary toxicity test:
All exposure groups: 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μg/mL

Main experiment:
4-hour exposure group (-S9): 0, 125, 250, 500, 750, 1000, 1500 and 2000 μg/mL.
4-hour exposure group (+S9): 0, 125, 250, 500, 750, 1000, 1500 and 2000 μg/mL.
24-hour exposure group (-S9): 0, 62.5, 125, 250, 500, 750, 1000, 1500 and 2000 μg/mL.

Vehicle / solvent:

The test item was soluble in water at 20 mg/mL in a solubility checks performed in-house, therefore water was used as the vehicle

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 µg/mL for 4-hour exposure

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

no

Positive controls:

yes

Remarks:

0.075 µg/mL for 24-hour continuous exposure

Positive control substance:

other: Demecolcine

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterile water

True negative controls:

no

Positive controls:

yes

Remarks:

5 µg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

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 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 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 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 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, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 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. The dose range of test item used was 0, 125, 250, 500, 750, 1000, 1500 and 2000 μg/mL.
i) 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. The dose range of test item used was 0, 125, 250, 500, 750, 1000, 1500 and 2000 μg/mL.
ii) 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, 62.5, 125, 250, 500, 750, 1000, 1500 and 2000 μg/mL.

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.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Statistics:

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.

Species / strain:

primary culture, other: whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 7.81 to 2000 μg/mL. The maximum dose was the maximum dose level.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level in any exposure group
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 2000 μg/mL in the all three exposure groups. The test item induced no evidence of toxicity in the 4-hour exposure groups but there was evidence of toxicity in the 24-hour continuous exposure group.
The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level for all exposure groups.

Micronucleus Test – Main Experiment
The qualitative assessment of the slides determined that there were binucleate cells suitable for scoring at the maximum dose level of test item in all three exposure groups.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level in any exposure group.
The CBPI data confirms that the qualitative observations in that no dose-related inhibition of CBPI was observed for the 4-hour exposure groups.
In the 24-hour continuous exposure group, 21%, 70% and 79% cytostasis was observed at 250, 500 and 1000 μg/mL, respectively. Above this dose level there were insufficient numbers of binucleates present for analysis. Therefore, the maximum dose level selected for binucleate cell analysis was limited by toxicity to 500 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.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation

The dose levels of the controls and the test item are given in the table below:

Group	Final concentration of test itemBayscript Magenta BB(µg/mL)
4-hour without S9	0*, 125, 250, 500, 750, 1000*, 1500*, 2000*, MMC0.2*
4-hour with S9 (2%)	0*, 125, 250, 500, 750, 1000*, 1500*, 2000*, CP5*
24-hour without S9	0*, 62.5*, 125*, 250*, 500*, 750, 1000, 1500, 2000,DC0.075*

*  = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC = Mitomycin C

CP = Cyclophosphamide

DC = Demecolcine

Conclusions:

The test item, Bayscript Magenta BB, 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:

Introduction

This report describes the results of an in vitro study 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 on toxicity or the maximum recommended dose level. The dose levels selected for the Main Test were as follows:

Group	Final concentration of test itemBayscript Magenta BB(µg/mL)
4-hour without S9	0, 125, 250, 500, 750, 1000, 1500, 2000
4-hour with S9 (2%)
24-hour without S9	0, 62.5, 125, 250, 500, 750, 1000, 1500, 2000

Results

All vehicle (sterile distilled water) 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 was non-toxic to human lymphocytes in the 4-hour exposure groups but was toxic to human lymphocytes in the 24-hour exposure group but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that either was the maximum recommended dose level or exceeded the optimum level of toxicity, depending on exposure group.

Conclusion

The test item, Bayscript Magenta BB was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Genetic toxicity in vivo

Description of key information

No data available

Justification for classification or non-classification

Bayscript Magenta BB is not mutagenic in vitro 1) in a bacterial test and 2) in aHPRTtest withV79 cells and was considered to be non-clastogenic and non-aneugenic to human lymphocytes