Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test Macrolex Rotviolett R was using Salmonella typhimurium LT2 mutants TA 1535, TA 100, TA 1537, TA 98 and TA 102. MACROLEX Rotviolett R was negative (non-mutagenic) without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

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

In an OECD guideline 487 (In vitro Mammalian Cell Micronucleus Test) study, Macrolex Rotviolet R did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells. Therefore, Macrolex Rotviolett R is considered to be negative (non-mutagenic) in this in vitro micronucleus test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
In an Ames test MACROLEX Rotviolett R was using Salmonella typhimurium LT2 mutants TA 1535, TA 100, TA 1537, TA 98 and TA 102.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Histidine deficient mutants - riginal strains were obtained from Prof. Bruce ames
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 16, 50, 158, 500, 1581 or 5000 µg/plate
0, 16, 50, 158, 500, 1581 or 5000 µg/tube
Vehicle / solvent:
DMSO (dimethylsulphoxide)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: 4-nitro-1,2-phenylene diamine, 2-aminoanthracene
Details on test system and experimental conditions:
MACROLEX Rotviolett R was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2
mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.
Additionally MACROLEX Rotviolett R was screened in an independent repeat using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 µg per tube after preincubation for 20 minutes at 37°C. All other conditions remained unchanged.
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, A 100 and TA 98 this increase should be about twice that of negative controls, whereas for A 1537, at
least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
Key result
Species / strain:
other: TA 98, TA 100, TA 102, TA 1535, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Plate incorporation test: Doses up to and including 500 µg per plate did not cause any bacteriotoxic effects. Preincubation test: Doses up to and including 1581µg per tube did not cause any bacteriotoxic effects.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Evidence of mutagenic activity of MACROLEX Rotviolett R was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.
Conclusions:
Negative
Executive summary:

In an Ames test MACROLEX Rotviolett R was using Salmonella typhimurium LT2 mutants TA 1535, TA 100, TA 1537, TA 98 and TA 102. MACROLEX Rotviolett R was negative (non-mutagenic) without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 14 July 2016 Experimental completion date 18 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian forward mutation assay
Specific details on test material used for the study:
Appearance: Dark violet powder
Storage Conditions: At room temperature
Expiry Date: 28 July 2017
Stability in Solvent: 200 mg/mL stable in DMSO for 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; 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 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: 15.6, 31.3, 62.5, 125.0, 250.0, 500.0, 1000.0 and 2000.0 µg/ml
The highest concentration was chosen with respect to the current OECD Guideline 476.

Relevant cytotoxic effects, indicated by a relative cloning efficiency of 50% or below were observed at 500.0 μg/mL and above 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 125 μg/mL and above after 4 hours treatment with and without 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 and without S9-mix: 7.8, 15.6, 31.3, 62.5, 125.0 and 250.0 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 DMSO. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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 (4-hour treatment time). The maximum concentration in the pre-test was 2000 μg/mL based on the solubility properties of the test item in 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 1.2×10^7 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 2.0×106 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 2.0×10^6 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 5×10^5 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 Rotviolett R 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 precipitation at the end of treatment was observed at 62.5 µg/mL and above with and without metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency 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 11.1 up to 16.4 mutants per 10^6 cells; the range of the groups treated with the test item was from 6.8 up to 20.7 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 experimental conditions reported Macrolex Rotviolett R did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Macrolex Rotviolett R is considered to be non-mutagenic in this HPRT assay.
Executive summary:

Summary

The study was performed to investigate the potential of Macrolex Rotviolett R 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 concentration of the pre-test on toxicity (2000 µg/mL) was chosen with respect to the current OECD Guideline 476. The dose range of the main experiment was limited by precipitation of the test item.

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 Rotviolett R did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Macrolex Rotviolett R is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 27 July 2016 Experimental completion date: 03 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Expiry Date: 28 July 2017
Storage Conditions: At room temperature
Appearance: Solid, violet
No correction for purity was made.
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate (doubling time of V79 cells in stock cultures: approximately 13 hours, determined on December 17, 2010) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 ± 1.

Large stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH. This allows the repeated use of the same cell culture batch in experiments. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Culture conditions
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 105 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.

Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. Per culture approximately 5.0 – 6.0 x 105 cells were seeded into 25 cm2 plastic flasks.

All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
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:
Experiment:
IA** and IA (4 hour exposure period, with and without S9-mix) - 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/ml
IB* and IB (4 hour exposure without S9-mix) - 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/ml
II** (24 hour exposure without S9-mix) - 5.9, 8.8, 13.2, 19.8, 29.6, 44.4, 66.7 and 100 µg/ml
II (24 hour exposure without S9-mix) - 0.5, 0.9, 1.6, 2.8, 4.9, 8.5, 14.9, 26.1, 45.7 and 80.0 µg/ml

* Was repeated due to invalid solvent control
** Was repeated due to strong cytotoxicity in the positive control

Initial doses were based on the OECD guideline and further doses were determined based on observations in the pre-experiment.
Vehicle / solvent:
Stock formulations of the test item and serial dilutions were made in DMSO. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
0.1 µg/ml
Positive control substance:
mitomycin C
Remarks:
Pulse treatment, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
6.0 µg/ml
Positive control substance:
other: Griseofulvin
Remarks:
Continuous treatment, without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
3.0 µg/ml
Positive control substance:
cyclophosphamide
Remarks:
Pulse treatment, with metabolic activation
Details on test system and experimental conditions:
The induction of cytogenetic damage in V79 cells (Chinese hamster cell line) was assessed in three independent experiments with one preparation interval (24 hours).

Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 11 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

Cytogenetic Experiment
Pulse exposure
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.

Continuous exposure (without S9 mix)
The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item. At the same time Cytochalasin B was added to the cell culture (1.5 µg/mL). The medium was not changed until preparation of the cells.

Preparation of cells
Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvest and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37°C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labelled with a computer-generated random code to prevent scorer bias.
Evaluation criteria:
A test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data

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 and/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.

However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.


Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test item Macrolex Rotviolett R, suspended (Exp. I) or dissolved (Exp. II) in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Three independent experiments were performed. In Experiment IA, the exposure period was 4 hours with S9 mix. In Experiment IB, the exposure period was 4 hours without S9 mix. In Experiment II, the exposure period was 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. At least 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined.
The highest treatment concentration in this study, 2000 µg/mL was chosen with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
Precipitation of the test item in the culture medium was observed at the end of treatment at:
Experiment IA (with S9): 31.3 µg/mL and above
Experiment IB (without S9): 31.3 µg/mL and above
Experiment II (without S9): 26.1 µg/mL and above
No relevant influence on osmolarity or pH was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.
In Experiment IB in the absence of S9 mix, statistically significant increases in the number of micronucleate cells, clearly within the range of the historical control data (0.0 – 2.62 % micronucleate cells), were observed after treatment with 7.8 and 15.6 µg/mL (2.15 and 2.10 %, respectively) and therefore declared as biologically not relevant.
In Experiment IA in the presence of S9 mix, one statistically significant increase in the number of micronucleate cells (2.30 %), above the range of the historical control data (0.0 –2.10 % micronucleate cells) was observed after treatment with 7.8 µg/mL. This can be declared as biologically irrelevant, because no significant increases were observed at higher concentration and consequently, the increase is not concentration-related.
In Experiment II in the absence of S9 mix after continuous treatment, no relevant increases in micronucleate cells were observed at any evaluated concentration (4.9, 8.5, 14.9 or 26.1 µg/mL).
Either Griseofulvin (6.0 µg/mL), MMC (0.1 µg/mL) or CPA (3.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Concentrations applied

Exp.

Prep.
interval

Exposure
period

Concentrations in µg/mL

Without S9 mix

IA**

24 hrs

 4 hrs

2.0

3.9

7.8

15.6

31.3P

62.5P

125P

250P

500P

1000P

2000P

IB*

24 hrs

 4 hrs

 

3.9

7.8

15.6

31.3P

62.5P

125P

250P

500P

1000P

2000P

IB

24 hrs

 4 hrs

 

3.9

7.8

15.6

31.3P

62.5P

125P

250P

500P

1000P

2000P

II**

24 hrs

24 hrs

 

 

 

5.9

8.8

13.2

19.8

29.6P

44.4P

66.7P

100P

II

24 hrs

24 hrs

 

0.5

0.9

1.6

2.8

4.9

8.5

14.9

26.1P

45.7P

80.0P

With S9 mix

IA

24 hrs

 4 hrs

2.0

3.9

7.8

15.6

31.3P

62.5P

125P

250P

500P

1000P

2000P

        Evaluated experimental points are shown in bold characters

P       Precipitation was observed microscopically at the end of treatment

*      Was repeated due to invalid solvent control

**    Was repeated due to strong cytotoxicity in the positive control

Summary of results

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

IB

24 hrs

Solvent control1

1.94

 

1.35

 

 

Positive control2

1.60

35.9

 5.70S

 

 

7.8#

1.90

4.8

 2.15S

 

 

15.6#

1.90

3.9

 2.10S

 

 

31.3P

1.86

8.5

1.20

Exposure period 24 hrs without S9 mix

II

24 hrs

Solvent control1

1.86

 

0.35

 

 

Positive control3

2.17

n.c.

7.00S

 

 

4.9

1.76

11.1

0.55

 

 

8.5

1.49

42.8

0.60

 

 

14.9

1.56

34.3

0.40

 

 

26.1P

1.49

43.3

0.70

Exposure period 4 hrs with S9 mix

IA

24 hrs

Solvent control1

1.95

 

1.45

 

 

Positive control4

1.55

41.6

 7.45S

 

 

7.8

1.76

19.6

 2.30S

 

 

15.6

1.68

28.4

2.10

 

 

31.3P

1.57

40.2

1.60

*      For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

#       The number of micronucleated cells was determined in a sample of 4000 binucleated cells

P       Precipitation occurred at the end of treatment

S       The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.  Not calculated as the CBPI is equal or higher than the solvent control value

1       DMSO                1.0 % (v/v)
2
           MMC                 0.1 µg/mL

3           Griseofulvin      6.0 µg/mL

4           CPA                    3.0 µg/mL

 

Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.
Therefore, Macrolex Rotviolett R is considered to be non-mutagenic in this in vitro micronucleus test.
Executive summary:

The test item Macrolex Rotviolett R, suspended (Exp. I) or dissolved (Exp. II) in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79 cells in vitro in three independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. IB

Exp. II

Exp. IA

Exposure period

 4 hrs

24 hrs

 4 hrs

Recovery

20 hrs

¾

20 hrs

Preparation interval

24 hrs

24 hrs

24 hrs

In each experimental group two parallel cultures were analyzed. Per culture at least 1000 cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (2000 µg/mL of the test item) was chosen with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In Experiment IB in the absence of S9 mix, statistically significant increases in the number of micronucleate cells, clearly within the range of the historical control data (0.0 – 2.62 % micronucleate cells), were observed after treatment with 7.8 and 15.6 µg/mL (2.15 and 2.10 %, respectively) and therefore declared as biologically not relevant.

In Experiment IA in the presence of S9 mix, one statistically significant increase in the number of micronucleate cells (2.30 %), above the range of the historical control data (0.0 –2.10 % micronucleate cells) was observed after treatment with 7.8 µg/mL. This can be declared as biologically irrelevant, because no significant increases were observed at higher concentration and consequently, the increase is not concentration-related.

In Experiment II in the absence of S9 mix after continuous treatment, no relevant increases in micronucleate cells were observed at any evaluated concentration (4.9, 8.5, 14.9 or 26.1 µg/mL).

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.


Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by thein vitromicronucleus test in Chinese hamster V79 cells.

Therefore, Macrolex Rotviolett R is considered to be non-mutagenic in this in vitro micronucleus test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Macrolex Rotviolet R was non-mutagenic (negative) in all in vitro tests. In a valid Ames test, in an HPRT assay according to OECD guideline 476 in V79 cells and an in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487 Macrolex Rotviolett R was negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.