1-hydroxy-4-(p-toluidino)anthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a guideline study according to OECD 471 using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA Macrolex Violet B was negative (non-mutagenic) without and with S9 mix in the plate incorporation test (experiment I) and the pre-incubation test (experiment II).

Several supporting Ames tests are available. In an Ames test using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA the test item was negative with and without metabolic activation. In an Ames test with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 strain TA 98 was negative with and without metabolic activation. In a published report Macrolex Violet B was tested for a mutagenic potential in the Salmonella reversion assay on strains  TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation in the plate incorporation and preincubation assay in dose up to 5000 µg/plate. Macrolex Violet B was negative with and without metabolic activation in TA 1535, TA 1538, TA 98 and TA 100. Macrolex Violet B was positive in TA 1537 without metabolic activation. The single positive result of TA 1537 in one test is regarded as not valid and maybe caused by impurities.

An Ames test with S. typhimurium TA 98 and TA 100 the test substance revealed a negative result. Overall, in the key study the Ames test was negative. The single positive result of TA 98 in one test is regarded as not valid and maybe caused by impurities.

Macrolex Violet B did not induce gene mutations in an HPRT assay according to OECD guideline 476 in in V79 cells of the Chinese hamster. Macrolex Violet B is considered to be negative (non-mutagenic) in this HPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

This study was performed to investigate the potential of Sandoplast Violet RSB to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment / Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity: Sandoplast Violet RSB
C.I.: Solvent Violet 13
Aggregate State at Room Temperature: Solid
Colour: Violet
Purity: 95.2 % (w/w)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment / Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Pre-Experiment for Toxicity
Ta evaluate the toxicity of the lest item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations were tested:
Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experimental Performance
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µI Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µI S9 mix (for lest with metabolic activation) or S9 mix substitution buffer (for lest without metabolic activation),
100 µI Bacteria suspension (cf. lest system, pre-culture of the strains),
2000µl Overlay agar
In the pre-incubation assay 100 µI test solution, 500 µI S9 mix / S9 mix substitution buffer and 100 µI bacterial suspension were mixed in a test tube and shaken at 37° C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Data Recording
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC

Acceptability of the Assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Rationale for test conditions:

In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I since no relevant toxic effects were observed and 5000 µg/plate were chosen as maximal concentration.

Evaluation criteria:

A test item is considered positive if a dose related increase in the number of revertants exceeding the threshold of twice or thrice the colony count of the corresponding solvent control is observed at more than one concentration. Furthermore, a biologically relevant and reproducible increase exceeding the threshold at one lest concentration is considered as positive.
A lest item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the lest points is considered non­ mutagenic in this system.

A biologically relevant response is described as follows:
An increase is considered relevant if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98, TA 100, and WP2 uvrA or thrice in strains TA 1535 and TA 1537 (3, 4).
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

A statistical analysis of the data is not mandatory.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sandoplast Violet RSB at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Conclusions:

Negative (test item did not induce gene mutations by base pair changes or frameshift in the genome of the strains used).

Executive summary:

The test item Sandoplast Violet RSB was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium  strains  TA 1535,  TA 1537,  TA 98,  and  TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment / Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:       33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the  test  item  showed  normal  background  growth  up  to  5000 µg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Sandoplast Violet RSB at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in- crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 11 February 2016 Experimental completion date 09 May 2016

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:

Appearance: Violet powder
Storage Conditions: At room temperature
Expiry Date: 02 June 2020
Stability in Solvent: Stable in THF over 4 and 24 hours

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.

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical 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 freezing, 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 2-3×10^6 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.

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 was used as metabolic activation system.

Test concentrations with justification for top dose:

Pre-experiment toxicity test
With and without S9-mix: 3.1, 6.3, 12.5, 25.0, 50.0, 100.0, 200.0 and 400.0 µg/ml
The maximum concentration in the pre-test was 400 µg/mL based on the solubility properties of the test item in THF and aqueous medium.

No relevant cytotoxic effect indicated by a relative cloning efficiency of 50% or below was observed up to the maximum concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Precipitation was noted at 100 μg/mL and above after 4 hours treatment without metabolic activation and at 50.0 µg/mL and above with metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Main experiment:
With S9-mix: 3.1, 6.3, 12.5, 25.0, 50.0 and 100.0 µg/ml
Without S9-mix: 6.3, 12.5, 25.0, 50.0, 100.0 and 200.0 µg/ml
The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Vehicle / solvent:

The vehicle was THF. The solvent was chosen for its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Remarks:

Final concentration: 300 μg/mL = 2.4 mM

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Final concentration: 2.3 μg/mL = 8.9 μM

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Pre-Test on Toxicity
A pre-test was performed in order to determine the toxicity of the test item. The maximum concentration in the pre-test was 400 µg/mL based on the solubility properties of the test item in THF and aqueous medium.
In addition the pH-value and the osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In the pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is evident as a reduction of the cloning efficiency (CE).

Experimental Performance
The experiment was performed with a treatment time of 4 hours with and without metabolic activation. Two parallel cultures were used throughout the assay.

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 "saline G".
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 x 10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of 7 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:

HPRT catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die.

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.

A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression analysis (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. However, both, biological and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item Macrolex Violett B 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.

In the main experiment turbidity or precipitation was observed at 100.0 µg/mL and above without metabolic activation and at 12.5 µg/mL and above with metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
No relevant and reproducible 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 up to the highest concentration with and without metabolic activation.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.
In the main experiment with and without S9 mix the range of the solvent controls was from 8.3 up to 16.3 mutants per 10^6 cells; the range of the groups treated with the test item was from 5.6 up to 23.2 mutants per 10^6 cells.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

In conclusion it can be stated that under the ex¬perimental conditions reported Macrolex Violett B did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Macrolex Violett B is considered to be non-mutagenic in this HPRT assay.

Executive summary:

Summary

The study was performed to investigate the potential of Macrolex Violett B 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 was based on the solubility properties of the test item and investigated in a pre-experiment. The concentration range of the main experiment was limited by precipitation of the test item.In the main experiment turbidity or precipitation was observed at 100.0 µg/mL and above without metabolic activation and at 12.5 µg/mL and above with metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.

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 Macrolex Violett B did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Macrolex Violett B is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

SOLVENT VIOLET 13 (Macrolex Violet B) was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with SOLVENT VIOLET 13. SOLVENT VIOLET 13 was not mutagenic (negative) in the micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Principles of method if other than guideline:

SOLVENT VIOLET 13 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.
Four groups each comprising 5 males, received an intraperitoneal injection. Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/ g body weight and one group was dosed with 500 mg/kg body weight. After dosing, the animals of the dose level of 2000 mg/kg body weight showed the following toxic signs: lethargy, rough coat and a hunched posture. The animals of the dose levels of 1000 and 500 mg/kg body weight showed no abnormalities after dosing, except two animals of the dose level of 500 mg/kg, which showed a rough coat.
A vehicle treated group served as negative control, a group treated with an intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.
Bone marrow of the groups treated with SOLVENT VIOLET 13 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

Identification: SOLVENT VIOLET 13

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Test System: NMRI BR mice (SPF), Recommended test System in international guidelines
Source: Charles Ri er, Sulzfeld, Germany.
Number of Animais per Test: 5 male mice per sampling time in each treatment group.
Age at Start of Treatment: Young adult animals were selected (6 weeks old).

Sex:

male

Details on test animals or test system and environmental conditions:

Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 15 air changes per hour, a temperature of 21± 3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day. Fluctuations from these optimal conditions were noted, but were considered not to have affected the integrity of the study.
Accommodation
Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material. Acclimatisation period was at least 5 days before statt of treatment under laboratory conditions.
Diet
Free access to Standard pelleted laboratory animal diet.
Water
Free access to tap-water.

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

The mice received an intraperitoneal injection of a maximum tolerated (high), an Intermediate and a low dose of SOLVENT VIOLET 13. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.
The dosing voiume was 10 ml/kg body weight.
The route and frequency of administration and the voiume administered of the negative and the positive control was the same as those of the test article.

Duration of treatment / exposure:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing SOLVENT VIOLET 13, 24 h after dosing of the vehicle and 48 h after dosing the positive contra!.

Frequency of treatment:

Single application

Post exposure period:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing SOLVENT VIOLET 13, 24 h after dosing of the vehicle and 48 h after dosing the positive contra!.

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Solvent Violet 13

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Solvent Violet 13

Dose / conc.:

500 mg/kg bw/day

Remarks:

Solvent Violet 13

Dose / conc.:

50 mg/kg bw/day

Remarks:

Cyclophosphamide

No. of animals per sex per dose:

5 male mice/dose

Control animals:

yes

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (Fresenius B.V., s-Hertogenbosch, The Netherlands) dosed at a single intraperitoneal injection of 50 mg salt/kg body weight.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing SOLVENT VIOLET 13, 24 h after dosing of the vehicle and 48 h after dosing the positive contra!. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell Suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.

Evaluation criteria:

ACCEPTABILITY OF ASSAY
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data ränge (mean + three times the Standard deviation): Males: 0.70%o ± 2.67%o indicated are means for n=80.

DATA EVALUATION AND STATISTICAL PROCEDURES
Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).
A test substance is considered negative in the micronucleus test if:
- one of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Micronucleus Test
Since there were no substantial differences between the sexes in toxicity after dosing the animals with 2000 mg/kg body weight, the micronuclues test was performed with male animals only. Based on the results of the dose range finding study, dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the micronucleus test. Five male animals were used in each treatment group.

Mortality and systemic toxic signs
The animals of the vehicle control group and the animals of the positive control group showed no abnormalities.

Micronucleated polvchromatic erythrocytes
The mean number of micronucleated polychromatic erythrocytes scored in SOLVENT VIOLET 13 treated groups were compared with the corresponding solvent control group. No biologically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of SOLVENT VIOLET 13 treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.
Cyclophosphamide, the positive control substance, induced a statisticaily significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, the acceptability criteria of the test were met.

Ratio polychromatic to normochromatic erythrocytes
The groups that were treated for 24 hours with SOLVENT VIOLET 13 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle Controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The dose group of 2000 mg SOLVENT VIOLET 13/kg body weight (48 hours treatment) and the group that was treated with cyclophosphamide showed a decrease in the ratio of olychromatic to normochromatic erythrocytes compared to the vehicle control.

CONCLUSION
It is concluded that this test is val d and that SOLVENT VIOLET 13 is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Conclusions:

Negative (not mutagenic in the micronucleus test under the experimental conditions).

Executive summary:

SOLVENT VIOLET 13 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Four groups each comprising 5 males, received an intraperitoneal injection. Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/ g body weight and one group was dosed with 500 mg/kg body weight. After dosing, the animals of the dose level of 2000 mg/kg body weight showed the following toxic signs: lethargy, rough coat and a hunched posture. The animals of the dose levels of 1000 and 500 mg/kg body weight showed no abnormalities after dosing, except two animals of the dose level of 500 mg/kg, which showed a rough coat.

A vehicle treated group served as negative control, a group treated with an intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with SOLVENT VIOLET 13 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with SOLVENT VIOLET 13.

The groups that were treated for 24 hours with SOLVENT VIOLET 13 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated for 48 hours with SOLVENT VIOLET 13 and the group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control.

It is concluded that SOLVENT VIOLET 13 is not mutagenic (negative) in the micronucleus test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In all valid in-vivo tests (Ames tests and HPRT assay) and the in-vivo Micronucleus Test (MNT) in mice, Macrolex Violet B was negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.