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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genetic toxicity: Rel 1, Key, Ames Test, OECD 471, 2012; non mutagenic.


In vitro genetic toxicity: Rel 2, Key, CAT, OECD 473, 1997; non clastogenic.


In vitro genetic toxicity: Rel 1, Key, HPRT test, OECD 476, 2012; non mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 3 July 1997 to 19 August 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
When comparing to the OECD TG 473 (1997 version), the following deviations were reported: - karyotype stability and absence of mycoplasme contamination were not reported; - cytotoxicity measurements are missing; - test-item confounding factors (e.g. pH, osmolality) are not mentioned; - historical control data are not available; - the exposure condition without S9Mix was 24 and 48 hrs instead of 3-6 and 24 hrs.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
yes
Remarks:
Exposure time without metabolic activation was 24 and 48 hrs, instead of 3-6 and 24 hrs. Reporting is not sufficient to assess the deviations (cf. rationale for reliability for further details).
Principles of method if other than guideline:
Chromosome aberration test with CHL/IU cells
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambiant temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: stable at ambient temperature
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: not reported
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: not reported
- Reactivity of the test material with the incubation material used (e.g. plastic ware): not reported

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): ultrasonication was conducted in the preparation of the test substance formulation
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: 50 mg/mL (highest concentration of test substance formulation)
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): suspension prepared using ultrasonication

FORM AS APPLIED IN THE TEST (if different from that of starting material): not applicable

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: none
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: CHL/IU obtained from Dainipon Pharmaceutical Co., Ltd.
- Suitability of cells: standard cell line as per OECD TG 473
- Normal cell cycle time (negative control): not reported
- Absence of Mycoplasma contamination: not reported
- Number of passages if applicable: 16
- Methods for maintenance in cell culture: plastic plate, 37°C, 5% CO2
- Cell cycle length, doubling time or proliferation index : doubling time ca. 15 hours
- Modal number of chromosomes: 25
- Periodically checked for karyotype stability: not reported
- Periodically ‘cleansed’ of spontaneous mutants: not reported


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's MEM, 10%. 37°C, 5% CO2.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Kikkoman Corporation (Lot No. RAA-363)
- method of preparation of S9 mix: Amount in 1 mL of S9 mix
S9 0,3 mL;
MgCl2 5 µmol;
KCl 33 µmol;
D-glucose 6-phosphate 5 µmol;
NADP 4 µmol;
Buffer (HEPES) 4 µmol;
Distilled water Added up to 1 mL.
- concentration or volume of S9 mix and S9 in the final culture medium: 5% S9 / 1.32 mg/mL S9 protein.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): not reported
Test concentrations with justification for top dose:
concentration [mg/ml] : 0.010, 0.020, 0.039, 0.078, 0.156, 0.313, 0.625, 1.250, 2.500, 5.000.
Based on the results of the preliminary test (no cytotoxicity).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 1% CMC-Na solutions

- Justification for choice of solvent/vehicle: Vehicle of the test substance was selected based on the result of a solubility test. In the solubility test, physiological saline and dimethyl sulfoxide were used as vehicles. As a result, the test substance was not dissolved in physiological saline at 50 mg/mL or in dimethyl sulfacide at 500 mg/mL. Te test substance suspended with ultrasonication in 1% CMC-Na solutions at 50 mg/mL. Base don these results, 1% CMC-Na solutions with which the test substance suspense in better condition could be prepared, was selected as the vehicle of the test substance.

- Justification for percentage of solvent in the final culture medium: 1%, based on preliminary solubility experiment. In line with OECD TG 473.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) duplicate
- Number of independent experiments: 3 (24-hrs -S9 / 48-hrs -S9 / 6-hrs + S9)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2x10E03 cells/well
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: n/a
- Exposure duration/duration of treatment: 24-hrs -S9 / 48-hrs -S9 / 6-hrs + S9
- Harvest time after the end of treatment (sampling/recovery times): 24-hrs -S9 / 48-hrs -S9 / 24-hrs + S9

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. Colcemid at 0.2 µg/mL, for 2 hours exposure.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): not reported
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): duplicate cultures. 200 cells per experiments.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The cells were fixed (10% formaline for 15 minutes), stained (0.1% crystal violet for 15 minutes), and extracted (acetic acid : 50% ethanol = 1:99) and the optical absorption (620 nm) was measured with a microplate reader.
- Determination of polyploidy: not reported
- Determination of endoreplication: not reported

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: cell growth index
- Any supplementary information relevant to cytotoxicity: none
Rationale for test conditions:
Selection of concentration was based on the result of the preliminary test. Because cytotoxicity was not recognised in the treatment with and without metabolic activation, 5 mg/mL was set as the highest concentration in each treatment condition. Five concentrations, by 4 step dilution with a common ratio of 2 from the highest concentration, were set.
Evaluation criteria:
Chromosomal aberration was classified as "gap", "break", "exchange", and "others" (fragmentation etc.) with chromatid type and chromosome type. A "gap" was defined as a clear achromatic region on an axis of a chromatid and the width of the achromatic

region was equal to or larger than the width of the chromatid.

In the chromosomal aberration test, judgment criteria described below were set for total incidence of aberrant cells. Data was shown in two ways; including and excluding "gap". The final judgment was conducted with data including "gap".

Negative (-): less than 5%
Inconclusive (±): 5% or more and less than 10%
Positive (+): 10% or more
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
cytotoxicity was not recognized in the treatment with and without metabolic activation, 5 mg/mL was set as the highest concentration in each treatment condition
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: not soluble
- Precipitation and time of the determination: not reported
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: not reported

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Number of chromosomal aberrant cells increased in the positive control group (MMC treatment group without S9 and B[a]P treatment with S9 group) and, therefore, these results showed that the test was conducted appropriately.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index. Not available
- Genotoxicity results (for both cell lines and lymphocytes): Number of chromosomal aberrant cells (structural aberrant cells and polyploid cells) did not increase in the treatment without or with metabolic activation. (cf. table of results attached to this ESR)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: not reported
Conclusions:
The test substance did not have the ability to induce chromosomal aberration under the test conditions.
Executive summary:

A Chromosome aberration test with CHL/IU cells was conducted with the test substance suspended in 1% CMC-Na at concentrations up to 5 mg/mL .


The number of chromosomal aberrant cells (structural aberrant cells and polyploid cells) did not increase in the treatment without or with metabolic activation. Number of chromosomal aberrant cells increased in the positive control group (MMC treatment group without S9Mix and B[a]P treatment group with S9Mix) and, therefore, these results showed that the test was conducted appropriately.


Based on the above results, it was judged that the test substance did not have the ability to induce chromosomal aberration.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13 September 2012 to 22 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD TG 476 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Principles of method if other than guideline:
This in vitro assay was performed to assess the potential of the test item to induce gene mutations by means of two independent HPRT experiments using the Chinese hamster cell line V79. The treatment time was 4 hours in the first experiment with and without metabolic activation. In the second experiment the cells were exposed to the test item for 24 hours without metabolic activation and 4 hours with metabolic activation.
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The analytical data verify that Macrolex Blau 3R is stable at room temperature for at least 4 hours.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: n/a
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING n/a
- Treatment of test material prior to testing (e.g. warming, grinding): suspended in DMSO
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution:

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: n/a
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni­ cal University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years.
- Normal cell cycle time (negative control): n/a

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: n/a
- Methods for maintenance in cell culture: stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use of the same cell culture batch in experiments.
- Cell cycle length, doubling time or proliferation index : doubling time 12-16 h, cloning efficiency of untreated cells > 50%
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
For seeding and treatment of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 pg/mL) and amphotericin B (1 %). For the selection of mutant cells the complete medium was supplemented with 11 pg/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:
Type and composition of metabolic activation system:
- source of S9: prepared by Harlan CCR
Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 were prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, weight approx. 220 - 320 g, Harlan Laboratories B.V., 5960 AD Horst, The Netherlands) induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg, Germany) and by peroral administrations of 80 mg/kg b.w. ß- naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules were kept at -20 °C for up to one week.
- method of preparation of S9 mix
An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 supernatant to reach following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate 4 mM NADP
in 100 mM sodium-phosphate-buffer, pH 7.4.
During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation was 26.9 mg/mL (Lot. No.: 060712) in the pre-experiment, 26.1 mg/mL (Lot. No.: 030812) in experiment I, and 48.7 mg/mL (Lot. No.: 150612) in experiment II.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo(a)pyrene.
Test concentrations with justification for top dose:
Experiment 1 / 4 hours / -S9: 9.8; 19.5; 39.0; 78.0p; 156.0p; 312.0p µg/mL.
Experiment 1 / 4 hours / + S9: 9.8; 19.5; 39.0p; 78.0p; 156.0p; 312.0p µg/mL.
Experiment 2 / 24 hours / -S9: 4.9; 9.8; 19.5; 39.0; 78.0 p; 156.0 p µg/mL.
Experiment 2 / 4 hours / +S9: 2.5; 4.9; 9.8; 19.5; 39.0 p; 78.0 p µg/mL.
p = precipitation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO 0.5 % (v/v)

- Justification for choice of solvent/vehicle: the same vehicle was used for the Ames test performed in the same CRO.

- Justification for percentage of solvent in the final culture medium: n/a
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5x10 at 5 cells were seeded into each flask with 15 mL of MEM (minimal essential medium)
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: n/a
- Exposure duration/duration of treatment: 4 hours (Exp. 1 and Exp. 2 with S9); 24 hours (Exp. 2 without S9).

FOR GENE MUTATION: n/a
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): Day 7
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days (expression time 7 days + incubation 8 days)
- Method used: flask
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure. 6-Thioguanine (11 µg/mL, about 8 days)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 500 cells per flask (2 flasks)
- Criteria for small (slow growing) and large (fast growing) colonies: not assessed

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: cloning efficiency
- Any supplementary information relevant to cytotoxicity: none

METHODS FOR MEASUREMENTS OF GENOTOXICIY n/a

- OTHER: n/a
Rationale for test conditions:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. The pre-experiment was performed using eight concentrations. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 pL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at sub-cultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the end of treatment by the unaided eye. Furthermore, test item induced changes in the osmolarity is considered.
The highest concentration used in the pre-test (1250 pg/mL) was chosen with regard to the solubility properties of the test item in aqueous media and DMSO. Test item concentrations between 9.78 pg/mL and 1250 pg/mL were used to evaluate toxicity in the presence and absence of metabolic activation (4 hours treatment).
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concen­ trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated 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 were considered together.
experimental group.
experiment I, culture I without S9 mix
experiment I, culture II without S9 mix experiment I, culture I with S9 mix experiment I, culture II with S9 mix experiment II, culture I without S9 mix experiment II, culture II without S9 mix experiment II, culture I with S9 mix experiment II, culture II with S9 mix
p-values 0.573 0.524 0.608 0.084 0.506 0.057 0.616 0.486
Key result
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: 7.35 vs 7.37 in solvent control
- Data on osmolality: 376 mOsm vs 387 in solvent control
- Possibility of evaporation from medium: not reported
- Water solubility: not reported. The highest concentration (1250 μg/mL) used in the range finding pre-experiment was limited by the solubility of the test item in DMSO and aqueous media
- Precipitation and time of the determination: The test medium was checked for precipitation or phase separation at the end of each treatment period prior to removal to the test item. Precipitation was observed at 39.1 µg/mL and above following 4 and 24 hour treatment with and without metabolic activation.
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: No relevant cytotoxic effect, indicated by a relative cloning efficiency below 50% was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: cf attached Table of Results
The highest solvent control value (37.0 colonies per 10E6 cells in experiment I, culture II with metabolic activation) slightly exceeded the range of historical solvent control data (3.4 - 36.6 mutant colonies/10E6 cells). The mean value of both parallel cultures however, (23.7 and 37.0 equal to 30.4 colonies per 10E6 cells) is fully acceptable. The cloning efficiency II of the solvent controls of the second culture with and without metabolic activation did not exceed 50% as required by the acceptance criteria. The data are valid however, as the solvent controls of the parallel cultures and the mean values of both parallel cultures (60%) are acceptable.
In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 10.6 up to 37.0 mutants per 10E6 cells; the range of the groups treated with the test item was from 4.9 up to 53.0 mutants per 10E6 cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: cf Tables of Results
- Genotoxicity results: cf. Tables of Results
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration.
In experiment I with metabolic activation the absolute value of the mutation frequency exceeded the range of the historical solvent control data (3.4 - 36.6 mutant colonies/10E6 cells) at 19.5 jg/mL (39.1 mutant colonies per 106 cells in culture I) and at 156.0 µg/mL (53.0 mutant colonies/10E6 cells in culture II). However, the threshold of three times the mutation frequency of the corresponding solvent control was neither reached nor exceeded at these data points.
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.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: cf Tables of Results
- Negative (solvent/vehicle) historical control data: cf Tables of Results

The highest concentration (1250 μg/mL) used in the range finding pre-experiment was limited by the solubility of the test item in DMSO and aqueous media. The dose range of the main experiments was limited by precipitation of the test item.


The tested concentrations are 9.8 - 312 µg/ml in experiment I and 2.5 - 168 µg/ml in experiment II.


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


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.

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

The study was performed to investigate the potential of Macrolex Blau 3R to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.


The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.


The highest concentration (1250 μg/mL) used in the range finding pre-experiment was limited by the solubility of the test item in DMSO and aqueous media. The dose range of the main experiments was limited by precipitation of the test item.


The tested concentrations are 9.8 - 312 µg/ml in experiment I and 2.5 - 168 µg/ml in experiment II.


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


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.


Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.


Therefore, Macrolex Blau 3R was negative in this HPRT assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 07 February 2012 to 07 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to the OECD TG 471. Note: 2-aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. However, each batch of S9 was checked prior to first use for its metabolising capacity by using reference mutagen(s).
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Principles of method if other than guideline:
Macrolex Blau 3R was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects, dissolved in DMSO, 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. The independent repeat experiment was performed as preincubation modification for 20 minutes at 37 °C in the absence of S9 mix in doses of up to and including 1600 µg per plate and in the presence of S9 mix of up to and including 5000 µg per plate.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: A stability test in the solvent did not reveal significant degradation of the active in­gredient.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: stable in the solvent at room temperature at concentrations ranging from 0.01 mg/mL to 500 mg/mL for at least four hours.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): suspended in DMSO
- Preliminary purification step (if any): none
- Final concentration of a dissolved solid, stock liquid or gel: n.a
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): not applicable

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: none
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: from the livers of at least 6 adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. The animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg bw, 5 days prior to sacrifice. The animals were prepared unfasted.
The livers were washed with cold (4 °C), 0.15 M KCI solution (approximately 1 ml KCI per 1 g liver), and then homogenized in fresh, cold (4 °C), 0.15 M KCI (approximately 3 ml KCI per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4 °C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80 °C in small portions.
- method of preparation of S9 mix:
70 mL of cofactors solution are composed as follows:
MgCl2 x 6 H2O 162.6 mg
KCI 246.0 mg
Glucose-6-phosphate, disodium salt 179.1 mg
NADP, disodium salt 315.0 mg
Phosphate buffer 100.0 mM
The S9 mix comprised 10 % S9 fraction, 70 % cofactor solution and 20 % 0.15 M KCI. The S9 fraction was derived from the preparation dated 16 NOV 2010 (protein content 21.8 mg per ml)
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9 fraction
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to first use, each batch was checked for its metaboliz­ ing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment 4 aliquots of the S9 mix were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37 °C. No in­ dication of contamination of S9 mix was found.
Test concentrations with justification for top dose:
Plate incorporation method: with and without S9 mix: 0, 50, 160, 500, 1600, 5000 µg/plate
Preincubation method: with S9 mix: 0, 50, 160, 500, 1600, 5000 µg/tube; without S9 mix 0, 16, 50, 160, 500, 1600 µg/tube (substance precipitation seen at 5000 µg/plate).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
- Justification for percentage of solvent in the final culture medium: not reported.
- Other: Macrolex Blau 3R formed blue suspensions in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
No "untreated" negative control was set up for the used solvent, since sufficient evi­ dence was available in the literature (e.g. Maron and Ames, 1983) and from historical control data, indicating that this solvent had no influence.
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
mitomycin C
other: 4-nitro-1,2-phenylene diamine, 2-nitrofluoren, 2-aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2 (plate incorporation and pre-incubation assays)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.2ml of thawed culture in 10mL nutrient broth.
- Test substance added: 1/ in agar (plate incorporation) and 2/ pre-incubation

TREATMENT SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C (pre-incubation assay)
- Exposure duration/duration of treatment: 48 hours at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: n/a

- OTHER:n/a
Rationale for test conditions:
In the plate incorporation assay concentrations up to 5000 µg/plate were assessed as per OECD TG 471. both in the presence and absence of metabolic activation.
In the liquid pre-incubation assay, concentrations up to 5000 µg/plate were assessed in the presence of metabolic activation. Due to substance's precipitation, doses were limited to 1600 µg/plate were evaluated in the absence of metabolic activation.
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, TA 100, TA 1537and TA 98 this in­crease should be about twice that of negative controls.
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: not reported
- Precipitation and time of the determination: Substance precipitation occurred in the plate and in the preincubation assay without S9 mix at the dose of 500 µg per plate and above and at the dose of 5000 µg per plate with S9 mix.
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: none

STUDY RESULTS - Table of results are attached to this ESR.
- Concurrent vehicle negative and positive control data: The positive controls sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluorene, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the sys­ tem's sensitivity and the activity of the S9 mix.

Ames test:
- Signs of toxicity: there was no indication of a bacteriotoxic effect of Macrolex Blau 3R at doses of up to and including 5000 pg per plate
- Individual plate counts - cf Table of results
- Mean number of revertant colonies per plate and standard deviation - cf. Table of results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: cf Table of results
- Negative (solvent/vehicle) historical control data: cf Table of results

The Salmonella/microsome plate incorporation test, employing doses of up to 5000 µg per plate, showed Macrolex Blau 3R not to produce bacteriotoxic effects. Substance precipitation occurred without S9 mix at 500 µg per plate and above and with S9 mix only at 5000 µg per plate.

Evaluation of individual dose groups, with respect to relevant assessment parame-ters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls.

In spite of the low doses used, positive controls increased the mutant counts to well over those of the negative controls, and thus demonstrated the system's high sensitivity.

Despite this sensitivity, no indications of mutagenic effects of Macrolex Blau 3R could be found at assessable doses of up to 5000 µg per plate in any of the Salmonella typhimurium strains.

Conclusions:
Macrolex Blau 3R is negative in the Salmonella/microsome test.
Executive summary:

Macrolex Blau 3R was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects, suspended in DMSO, at 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. The independent repeat experiment was performed as preincubation modification for 20 minutes at 37 °C in the absence of S9 mix in doses of up to and including 1600 µg per plate and in the presence of S9 mix of up to and including 5000 µg per plate. Other conditions remained unchanged.


 


Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred without S9 mix at the dose of 500 µg per plate and above and with S9 mix at 5000 µg per plate.


 


Evidence of mutagenic activity of Macrolex Blau 3R was not seen. No biologically relevant increase in the mutant count, in comparison to the negative controls, was observed in any of the strains tested, without and with S9 mix, in the plate incorporation as well as in the preincubation modification, under the experimental conditions applied.


 


The positive controls sodium azide, 4-nitro-1,2-phenylene diamine, 2-nitrofluorene, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.


 


Therefore, Macrolex Blau 3R is negative in the Salmonella/microsome test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6.1/1 : Summary of genotoxicity tests:


 










































Test n°



Test Guideline / Reliability



Focus



Strains  / cells tested



Metabolic activation



Test concentration



Statement



1 (2012)



Ames Test


(OECD 471, GLP)


K, rel.1



Gene mutation



S. thyphimurium


TA 1535,


TA 1537,


TA 98,


TA 100,


TA 102



-S9


+S9



Tested up to 5000 µg/plate in DMSO



-S9: not mutagenic


+ S9: not mutagenic



2 (1997)



CAT


(OECD 473, GLP)


K, rel.2



Chromosomal aberration



Chinese Hamster lung CHL/IU



-S9


+S9



Tested up to 5 mg/mL in 1% CMC-Na solutions



-S9: not clastogenic


+ S9: not clastogenic



3 (2012)



HPRT


(OECD 476, GLP)


K, rel.1



Gene mutation



Chinese Hamster lung V79



-S9


+S9



Tested up to 312 µg/mL in DMSO (limited by precipitation)



-S9: not mutagenic


+ S9: not mutagenic



Gene mutation Assay (Test n° 1 & 3)


A bacterial reverse mutation assay (Ames test) was performed with the substance (Test n°1). This study was used to conclude on the potential of the substance to induce gene mutation in bacteria.


No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains with any dose of test material, either in the presence or absence of metabolic activation, up to limit concentration. The test indicates that the substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test. 


Inability to produce gene mutation was confirmed using an in vitro forward mutation assay in Chinese Hamster V79 cells(Test n°3). None of the dose levels up to the precipitating concentration, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat tests. The test material did not induce forward mutations at the hprt locus in V79 mouse cells under activation and non activation conditions whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. The test material is therefore considered as negative for inducing forward mutations at the hprt locus in V79 mouse cells under activation and non-activation conditions used in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the test material to mammalian cells.


 


Chromosomal aberration (Test n°2)


The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in Chinese hamster lung cells, which measures the potential of a substance to increase the incidence the of structural chromosome aberrations in cultured Chinese hamster lung cells. None of the dose levels up to the limit concentration with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of two experiments, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells. The substance is therefore considered as negative for inducing chromosomal mutations in Chinese hamster lung cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonised classification: 


The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP).


 


Self-classification: 


Based on the available information, no additional classification is proposed according to the CLP and the GHS.