Registration Dossier
Registration Dossier
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EC number: 252-650-1 | CAS number: 35636-63-6
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Interpretation of results: negative
In conclusion, it can be stated that during the described mutagenicity test, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
The test substance did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2021
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Cytokinesis block (if used):
- Colchicine was used as the spindle inhibitor at 0.2 μg/mL
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9 homogenate was used as the metabolic activation system. The S9 homogenate was prepared from male Wistar rats induced with a single intraperitoneal injection of Aroclor 1254 (0.7 mL/rat ready to use solution), 5 days prior to sacrifice.
The S9 homogenate batch prepared and stored in the test facility either at -68 to -86 °C or in liquid
nitrogen until its use. Each batch of S9 homogenate is assessed for sterility, protein content (modified Lowry Assay, Sword and Thomson, 1980) and for its ability to metabolize the pro-mutagens 2-Aminoanthracene and Benzo (a) pyrene to mutagens using Salmonella typhimurium TA100 strain.
S9 homogenate was thawed immediately before use and mixed with the co-factor solution containing 4 mM NADP, 5 mM Glucose-6-phosphate, 8 mM MgCl2 and 33 mM KCl in PBS.
7.2.1 S9 Mix
The co-factor solution was prepared by dissolving the following co-factors in 18 mL PBS for the preliminary cytotoxicity test and the chromosome aberration assay.
NADP (4 mM) 57 mg
Glucose-6-phosphate (5 mM) 31 mg
Magnesium chloride (8 mM) 29 mg
Potassium chloride (33 mM) 44 mg
The S9 mix was prepared by mixing 4 mL S9 homogenate with 16 mL of the co-factor solution, for the preliminary cytotoxicity test and the chromosomal aberration assay. - Test concentrations with justification for top dose:
Test Concentrations
Following concentrations of the test item were used in the preliminary cytotoxicity test:
a) 7.81, b) 15.63, c) 31.25, d) 62.5, e) 125, f) 250, g) 500, h) 1000 and i) 2000 μg/mL (limit concentration as per OECD 473).
At the beginning and end of 3-hour exposure period, there was no precipitation of the test item, both in the presence and absence of metabolic activation at the test concentrations of 7.81, 15.63, 31.25, 62.5 and 125 µg/mL.
At the beginning and end of the 3-hour exposure, both in the presence and absence of metabolic activation, there was slight precipitation of the test item at 250 µg/mL and moderate precipitation of the test item at 500, 1000 and 2000 µg/mL.
Based on the guideline requirement, the test concentrations which exhibited moderate precipitation of the test item were not processed further and 250 µg/mL, which exhibited slight precipitation of the test item was selected as the top concentration in the preliminary cytotoxicity test.
Based on observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
Main test
Based on the observations of the preliminary cytotoxicity test, following concentrations of the test item are selected for testing in the chromosomal aberration assay
a) 15.63 b) 62.5 and c) 250 μg/mL- Vehicle / solvent:
- Sterile water
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 63830396) with a doubling time approximately 12 -14 hours and modal chromosome number of 20 was used as the test system.
The cell line was tested for mycoplasma in the testing facility. The karyotype analysis of this cell line was periodically performed and documented.
Cells were grown in T- 25 cm2 and T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air)
The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic
activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.
The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.
In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55 ± 5 % of the concurrent vehicle control) up to the highest tested concentration of 250 μg/mL, both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).
Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 μg/mL in the presence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250 μg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.
At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.
A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations. - Rationale for test conditions:
- Conditions according to guideline
- Evaluation criteria:
- Metaphases of all concentrations of the test item, the positive and vehicle control cultures were scored.
8.1 Definition for Aberrations
The chromosome and chromatid aberrations observed have been grouped into three categories - gaps, breaks (includes deletions and displacements) and exchanges.
Gap
Chromatid gap is a non-staining region of a single chromatid in which there is a minimal misalignment of the chromatid.
Chromosome gap is a non-staining region at the same locus in both chromatids of a single chromosome in which there is minimal misalignment of the chromatids.
Break
Chromatid break is discontinuity of a single chromatid in which there is clear misalignment of one of the chromatids.
Chromosome break is discontinuity at the same locus in both chromatids of a single chromosome, giving rise to an acentric fragment.
A chromatid deletion occurs when a bit of chromatid is missing as a result of a break.
A chromosome deletion occurs when bits of both chromatids are missing as a result of a break.
Chromatid displacement occurs when the fragment of the chromatid beyond the break point is not in alignment with the chromosome of origin.
Chromosome displacement occurs when the fragments of both chromatids beyond the break point are not in alignment with the chromosome of origin.
Exchange
Exchanges occur as a result of two or more chromatid lesions and the subsequent rearrangement of chromatid material.
Ring chromosomes are formed when a chromosome undergoes two breaks and the broken ends of the chromosome reunite in a ring structure either with or without a centromere.
Pulverization
In cells with pulverization, the chromosomes are reduced to masses of fragments. This represents the degree of damage inflicted on the chromosome. This aberration indicates the cytotoxic effect of the test item.
Polyploidy
Cell containing more than the diploid number (2n) of chromosomes, - Statistics:
- Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fischer exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- The test item, C.I. Pigment Yellow 175 was not clastogenic in CHO-K1 cells at the tested concentration and under the conditions of testing employed.
- Executive summary:
The clastogenic potential of the test item, C.I. Pigment Yellow 175 to induce chromosomal aberrations in mammalian cells was evaluated using cultured Chinese Hamster Ovary (CHO-K1) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).
The study consisted of a preliminary cytotoxicity test and a chromosome aberration assay. Chromosome aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.
The test item formed a free flowing suspension in sterile water at 200 mg/mL and a pasty suspension in DMSO at 200 mg/mL. Hence, sterile water was selected as the vehicle of choice for the chromosomal aberration test.
In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item did not exhibit the required level of cytotoxicity (reduction in the cell growth by 55± 5 % of the concurrent vehicle control)up to the highest tested concentration of 250µg/mL,both, in the presence and absence of metabolic activation (short term treatment) and in the absence of metabolic activation (long term treatment).
Based on these observations and preliminary cytotoxicity test results, it was decided to test up to a maximum of 250 µg/mL in thepresence and absence of metabolic activation with the 3-hour exposure and in the absence of metabolic activation with the 21-hours exposure period in the chromosomal aberration assay.
In the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate at the concentrations of 15.63, 62.5 and 250mg/mL in Experiments 1, 2 and 3 of the chromosomal aberration assay. Concurrent vehicle (SW) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation at 21 hour exposure) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at approximately 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.
At the highest concentrations tested, the reduction in cell growth as RICC was 47, 43 and 53 % in experiments 1, 2 and 3, respectively, compared to the vehicle control.
A total of 300 metaphases each from the SW control, each treatment level and the positive controls were evaluated for chromosomal aberrations.
There was no evidence of statistically significant induction of chromosomal aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.
The study indicated that the test item, C.I. Pigment Yellow 175 was not clastogenic at the concentrations tested and under the conditions of testing
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 29 AUG 2012 to 24 SEP 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD 471, GLP-compliant)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- E. coli WP2 uvr A
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 (induced with phenobarbital/beta-naphtoflavone; experiment I); hamster liver S9 (non-induced; experiment II)
- Test concentrations with justification for top dose:
- Experiment I: 3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: sodium azide (TA 1535, TA 100) / 4-Nitro-o-phenylene-diamine (TA 1537, TA 98) / methyl methane sulfonate (WP2 uvrA)
- Remarks:
- Without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, TA 98, WP2 uvrA)
- Remarks:
- with metabolic activation (rat liver S9 mix)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (TA 1535, TA 100, TA 1537, WP2 uvrA) / congo red (TA 98)
- Remarks:
- with metabolic activation (hamster liver S9 mix)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar, plate incorporation (Experiment I), preincubation (Experiment II);
DURATION
- Preincubation period: 1 hour
- Exposure duration: 72 hours
NUMBER OF REPLICATIONS: 3 plates
DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.
The bacteria used in this assay do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major draw¬back an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
8.4.1 Rat Liver S9 (Preparation by Harlan CCR)
Phenobarbital/b-naphthoflavone induced rat liver S9 will be used as the metabolic activation system. The S9 is 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 b-naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at –80 °C. Small numbers of the ampoules can be kept at –20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo[a]pyrene.
The protein concentration in the S9 preparation was 39.5 mg/mL (lot no. R 260412).
8.4.2 Hamster Liver S9 (Preparation by Harlan CCR)
The S9 liver microsomal fraction was prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters.
After decapitation of the anaesthetised animals the livers of the animals was removed, washed in 0.1 M sodium phosphate buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in deionised water and homogenised. The homogenate, diluted 1+3 in sodium phosphate buffer was centrifuged at 9,000 g for 25 minutes at 4 °C. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as congo red.
The protein concentration in the S9 preparation was 24.0 mg/mL (lot no. H 020712).
8.4.3 Rat S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-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.
8.4.4 Hamster S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution. The amount of S9 supernatant was 30% v/v. The concentrated cofactor solution yields the following concentrations in the S9 mix:
8.0 mM MgCl2
33.0 mM KCl
20.0 mM Glucose-6-phosphate
2.8 units/ml Glucose-6-phosphate-dehydrogenase
4.0 mM NADP
2.0 mM NADH
2.0 mM FMN
in 100 mM Sodium-Ortho-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. and Prival and Mitchell .
- Evaluation criteria:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant. - Statistics:
- According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
- Key result
- Species / strain:
- other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate in both experiments. Precipitation of the test item on the incubated agar plates was observed from 1000 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording. .
COMPARISON WITH HISTORICAL CONTROL DATA: performed
ADDITIONAL INFORMATION ON CYTOTOXICITY:
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 metabol¬ic activation. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results:
negative
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.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay. - Executive summary:
This study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and the Escherichia coli strain WP2 uvrA.
The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments: 3; 10; 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 metabolic activation in both independent 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 the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix).
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 2 NOV 2004 to 27 DEC 2004
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study (OECD TG 476)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
RPMI 1640 (Hepes buffered medium) supplemented with 15% horse serum, penicillin/streptomycin (100 U/mL respectively 100 µl/mL), 220 µg/mL sodium pyruvate and 1.25 U/mL Amphotericin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 (induced with phenobarbital/beta-naphtoflavone)
- Test concentrations with justification for top dose:
- Experiment I:
without S9 mix: 11.7; 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
with S9 mix: 23.4; 46.9; 93.8; 187.5, and 375.0 microgram/mL
Experiment II: without S9 mix: 11.7; 23.4; 46.9; 93.8; and 187.5 microgram/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the cell cultures - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- without metabolic activation
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
DURATION
- Exposure duration: 4 h (Experiment I: with and w/o metabolic activation) and 24 h (Experiment II w/o metabolic activation)
- Expression time (cells in growth medium): 3 days total (i.e. experiment I: 4 hours treatment period plus 68 hours expression time; experiment II: 24 hours treatment period plus 48 hours expression time)
- Selection time (if incubation with a selection agent): 10 to 15 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT)
NUMBER OF REPLICATIONS: two independent experiments, using two parallel cultures each
NUMBER OF CELLS USED: Per culture 1 x 10EXP7 cells
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth; other: relative suspension growth - Evaluation criteria:
- A test item is classified as positive if it induces either a reproducible concentration-related increase in the mutant frequency or a reproducible positive response for at least one of the test points.
A test item producing neither a reproducible concentration-related increase in the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test item is classified as mutagenic if it reproducibly induces a mutation frequency that is at least two times higher than the spontaneous mutation frequency (negative or solvent control) in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth and/or cloning efficiency 1 (survival of the cells after treatment) is less than 10 % of the solvent control or the cloning efficiency 2 (viability) after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated. - Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- indicated by reduced relative cloning efficiency 1 in experiment I at 375 µg/mL in both cultures
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- experiment I: 1st culture at 375 µg/mL, 2nd culture at 187.5 µg/mL and above; experiment II: both cultures at 187.5 µg/mL and above
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Precipitation of the test item was observed by the unaided eye at 187.5 and 375.0 microgram/mL in all experimental parts.
COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequency did not exceed the historical range of negative and solvent controls at any concentration with and without metabolic activation. The threshold of two times the number of mutant colonies/10EXP6 cells was reached at 46.9 microgram/mL in the second culture of experiment I (with S9 mix). Since this effect was not observed at higher concentrations or in the parallel culture, this isolated effect was judged as biologically irrelevant.
The mutation frequency of the positive control in the first culture of experiment I fell just short of the historical range of positive controls with 233 versus 292 colonies per 10EXP6 cells. Since the threshold of twice the mutation frequency of the solvent control was clearly exceeded and the mean value of both positive controls is well within the historical range, this effect was considered as irrelevant fluctuation.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I without metabolic activation relevant toxicity (relative cloning efficiency 1 and / or relative total growth of less than 50 % survival) was detected at 187.5 µg/mL and above in the second culture. In culture I a clear toxic effect occurred at the highest concentration of 375 µg/mL.
In experiment I with metabolic activation a toxic effect indicated by a reduced relative cloning efficiency 1 was observed at 375 µg/mL in both cultures.
In the second experiment (24 h treatment without metabolic activation) relevant toxic effects occurred at 187.5 µg/mL and above in both cultures. - Remarks on result:
- other: all strains/cell types tested
- Conclusions:
- Interpretation of results (migrated information):
negative
The test substance did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test. - Executive summary:
Mouse Lymphoma cells (L5178Y TK+/-) were treated with the test substance at five or six concentrations in the range of 11.7 to 375 microgram/mL. Precipitation of the test substance was detectable at dose level 187.5 and 375 microgram/mL. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, both in the presence or absence of metabolic activation, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.
Referenceopen allclose all
TABLE 3. Results of Preliminary Cytotoxicity Test
Treatment (µg/mL) | Presence of metabolic activation (3-hour exposure) | Absence of metabolic activation (3-hour exposure) | Absence of metabolic activation (21-hour exposure) | ||||||
Final – initial cell count (1x106/flask) | Cell growth index RICC (%) | Cell growth inhibition (%) | Final – initial cell count (1x106/flask) | Cell growth index RICC (%) | Cell growth inhibition (%) | Final – initial cell count (1x106/flask) | Cell growth index RICC (%) | Cell growth inhibition (%) | |
Sterile water | 0.78125 | 100 | 0 | 0.83125 | 100 | 0 | 0.71875 | 100 | 0 |
7.81 | 0.71875 | 92 | 8 | 0.79375 | 95 | 5 | 0.63125 | 88 | 12 |
15.63 | 0.63125 | 81 | 19 | 0.73125 | 88 | 12 | 0.55625 | 77 | 23 |
31.25 | 0.56875 | 73 | 27 | 0.68125 | 82 | 18 | 0.50625 | 70 | 30 |
62.5 | 0.51875 | 66 | 34 | 0.60625 | 73 | 27 | 0.43125 | 60 | 40 |
125 | 0.48125 | 62 | 38 | 0.55625 | 67 | 33 | 0.38125 | 53 | 47 |
250 | 0.41875 | 54 | 46 | 0.49375 | 59 | 41 | 0.31875 | 44 | 56 |
Base line cell count: 1.58125 X 106/flask
TABLE 4. Summary Results of Chromosomal Aberration Assay - Experiment 1
Treatment (µg/mL) | No. of metaphases scored | No. (%) of metaphases with aberrations@ | Total No. (%) of aberrant metaphases* | Cell Growth Inhibition (%) | ||||||
Gaps | Breaks | Exchanges | Includi ng Gaps | Excluding Gaps | ||||||
Cs | Ct | Cs | Ct | Cs | Ct | |||||
Sterile water | 300 |
0 |
0 |
1 (0.33) |
0 |
0
|
0 |
1 (0.33) |
1 (0.33) | 0 |
15.63 | 300 |
0
|
0 |
0 |
0 |
0 |
0 |
0 |
0 | 16 |
62.5 | 300 |
0
|
0 |
0 |
0 |
0 |
0 |
0 |
0 | 33 |
250 | 300 |
0 |
0 |
1 (0.33) |
0 |
0 |
0 |
1 (0.33) |
1 (0.33) | 47 |
CPA 55 | 300 |
1 (0.33) |
0 |
12 (4.00) |
19 (6.33) |
38 (12.67) |
31 (10.33) |
73 (24.33) |
72+ (24.00) | 35 |
*: Metaphase plate with one or more than one aberration considered as one metaphase plate with aberrations
@: values are the sum of two replicates and values in parenthesis represent %
Cs: Chromosome type Ct: Chromatid type
CPA: Cyclophosphamide monohydrate +: Significantly higher than control (p <0.05) by Fischer exact test Note: There were no incidences of polyploidy and endoreduplicated cells
TABLE 5. Summary Results of Chromosomal Aberration Assay - Experiment 2
Treatment (µg/mL) | No. of metaphases scored | No. (%) of metaphases with aberrations@ | Total No. (%) of aberrant metaphases* | Cell Growth Inhibition (%) | ||||||
Gaps | Breaks | Exchanges | Including Gaps | Excluding Gaps | ||||||
Cs | Ct | Cs | Ct | Cs | Ct | |||||
Sterile water | 300 |
1 (0.33) | 0 | 0 | 0 | 0 | 0 |
1 (0.33) | 0 | 0 |
15.63 | 300 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
10 |
62.5 | 300 | 0 |
1 (0.33) | 0 | 0 | 0 | 0 |
1 (0.33) | 0 |
25 |
250 | 300 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
43 |
*: Metaphase plate with one or more than one aberration considered as one metaphase plate with aberrations
@: values are the sum of two replicates and values in parenthesis represent %
Cs: Chromosome type Ct: Chromatid type
Note: There were no incidences of polyploidy and endoreduplicated cells
TABLE 6. Summary Results of Chromosomal Aberration Assay - Experiment 3
Treatment (µg/mL) | No. of metaphases scored | No. (%) of metaphases with aberrations@ | Total No. (%) of aberrant metaphases* | Cell Growth Inhibition (%) | ||||||
Gaps | Breaks | Exchanges | Including Gaps | Excluding Gaps | ||||||
Cs | Ct | Cs | Ct | Cs | Ct | |||||
Sterile water | 300 | 0 | 0 |
1 (0.33) | 0 | 0 | 0 |
1 (0.33) |
1 (0.33) | 0 |
15.63 | 300 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 22 |
62.5 | 300 | 0 | 0 |
0 |
1 (0.33) | 0 | 0 |
1 (0.33) |
1 (0.33) | 38 |
250 | 300 | 0 | 0 |
1 (0.33) | 0 | 0 | 0 |
1 (0.33) |
1 (0.33) | 53 |
EMS 600 | 300 |
4 (1.33) | 0 |
35 (11.67) |
36 (12.00) |
9 (3.00) |
15 (5.00) |
84 (28.00) |
81+ (27.00) | 28 |
*: Metaphase plate with one or more than one aberration considered as one metaphase plate with aberrations
@: values are the sum of two replicates and values in parenthesis represent %
Cs: Chromosome type Ct: Chromatid type
EMS: Ethyl methanesulfonate +: Significantly higher than control (p <0.05) by Fischer exact test
Note: There were no incidences of polyploidy and endoreduplicated cells
ANNEXURE 2. Historical Vehicle and Positive Control Data
Test System: CHO cells No. of Studies: 56
Vehicle Control
Parameter | Total No. of Metaphases with Aberrations Excluding Gaps | |
Presence of Metabolic Activation | Absence of Metabolic Activation | |
Range | 0 - 5 | 0 - 4 |
Mean ± SD | 0 ± 1 | 1 ± 1 |
Positive Control
Parameter | Total No. of Metaphases with Aberrations Excluding Gaps | |
Presence of Metabolic Activation | Absence of Metabolic Activation | |
Range | 51 - 204 | 63 - 248 |
Mean ± SD | 124 ± 37 | 121 ± 32 |
CHO: Chinese Hamster Ovary Cells
SD: Standard deviation
1.1 Pre-Experiment (= Experiment I)
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
||||||
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
|
|
Without Activation |
DMSO |
|
|
15 ± 5 |
12 ± 2 |
26 ± 8 |
142 ± 8 |
57 ± 16 |
Untreated |
|
|
13 ± 1 |
14 ± 6 |
29 ± 6 |
146 ± 9 |
58 ± 11 |
|
Test item |
3 µg |
|
15 ± 6 |
12 ± 3 |
24 ± 3 |
137 ± 16 |
56 ± 15 |
|
10 µg |
|
17 ± 6 |
12 ± 3 |
22 ± 1 |
137 ± 13 |
54 ± 4 |
||
|
33 µg |
|
17 ± 5 |
12 ± 5 |
26 ± 4 |
150 ± 3 |
55 ± 9 |
|
|
100 µg |
|
12 ± 3 |
13 ± 0 |
21 ± 4 |
137 ± 15 |
62 ± 12 |
|
|
333 µg |
|
15 ± 5 |
15 ± 1 |
23 ± 3 |
143 ± 18 |
67 ± 7 |
|
|
1000 µg |
|
13 ± 4P M |
14 ± 1P M |
25 ± 4P M |
137 ± 10P M |
53 ± 6P M |
|
|
2500 µg |
|
11 ± 2P M |
16 ± 4P M |
29 ± 4P M |
150 ± 16P M |
54 ± 8P M |
|
|
5000 µg |
|
11 ± 4P M |
16 ± 4P M |
32 ± 5P M |
142 ± 23P M |
57 ± 10P M |
|
NaN3 |
10 µg |
|
1908 ± 67 |
|
|
1913 ± 86 |
|
|
4-NOPD |
10 µg |
|
|
|
289 ± 9 |
|
|
|
4-NOPD |
50 µg |
|
|
71 ± 14 |
|
|
|
|
MMS |
3.0 µL |
|
|
|
|
|
1170 ± 110 |
|
|
|
|
|
|
|
|
|
|
With Activation |
DMSO |
|
|
23 ± 4 |
25 ± 5 |
41 ± 3 |
145 ± 17 |
67 ± 4 |
Untreated |
|
|
17 ± 4 |
22 ± 2 |
39 ± 8 |
170 ± 14 |
73 ± 10 |
|
Test item |
3 µg |
|
22 ± 6 |
21 ± 1 |
46 ± 8 |
148 ± 7 |
71 ± 6 |
|
10 µg |
|
23 ± 6 |
23 ± 3 |
36 ± 8 |
165 ± 11 |
67 ± 6 |
||
|
33 µg |
|
27 ± 1 |
25 ± 1 |
37 ± 5 |
141 ± 17 |
73 ± 11 |
|
|
100 µg |
|
22 ± 4 |
27 ± 1 |
34 ± 4 |
147 ± 5 |
74 ± 6 |
|
|
333 µg |
|
21 ± 1 |
23 ± 4 |
34 ± 4 |
139 ± 8 |
76 ± 8 |
|
|
1000 µg |
|
24 ± 2P |
23 ± 4P |
23 ± 3P |
127 ± 4P |
60 ± 4P |
|
|
2500 µg |
|
22 ± 2P M |
21 ± 3P M |
22 ± 2P M |
143 ± 6P M |
51 ± 5P M |
|
|
5000 µg |
|
16 ± 3P M |
19 ± 4P M |
19 ± 4P M |
140 ± 14P M |
54 ± 6P M |
|
2-AA |
2.5 µg |
|
357 ± 16 |
327 ± 13 |
1851 ± 30 |
2716 ± 505 |
|
|
2-AA |
10.0 µg |
|
|
|
|
|
261 ± 28 |
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
P M |
Precipitate Manual count |
1.2 Experiment II
Metabolic Activation |
Test Group |
Dose Level (per plate) |
|
Revertant Colony Counts (Mean ±SD) |
||||
|
|
|
||||||
|
|
|
TA 1535 |
TA 1537 |
TA 98 |
TA 100 |
WP2 uvrA |
|
|
|
|
|
|
|
|
|
|
Without Activation |
DMSO |
|
|
21 ± 3B M |
19 ± 2 |
27 ± 6 |
125 ± 12 |
49 ± 8 |
Untreated |
|
|
17 ± 4B M |
21 ± 1 |
28 ± 7 |
161 ± 4 |
49 ± 7 |
|
Test item |
3 µg |
|
20 ± 2B M |
20 ± 3 |
23 ± 1 |
128 ± 35 |
55 ± 7 |
|
10 µg |
|
22 ± 4B M |
22 ± 6 |
22 ± 2 |
125 ± 5 |
57 ± 9 |
||
|
33 µg |
|
25 ± 3B M |
21 ± 2 |
27 ± 4 |
122 ± 13 |
60 ± 7 |
|
|
100 µg |
|
24 ± 2B M |
19 ± 7 |
22 ± 2 |
128 ± 6 |
50 ± 7 |
|
|
333 µg |
|
24 ± 4B M |
20 ± 4 |
20 ± 3 |
138 ± 13 |
51 ± 3 |
|
|
1000 µg |
|
22 ± 5B M P |
21 ± 3P |
23 ± 1P |
115 ± 6P |
36 ± 6P |
|
|
2500 µg |
|
17 ± 2B M P |
24 ± 7P M |
37 ± 2P M |
117 ± 11P M |
40 ± 8P M |
|
|
5000 µg |
|
21 ± 3P M B |
28 ± 2P M |
42 ± 8P M |
111 ± 7P M |
41 ± 7P M |
|
NaN3 |
10 µg |
|
1086 ± 44B M |
|
|
2019 ± 211 |
|
|
4-NOPD |
10 µg |
|
|
|
280 ± 2 |
|
|
|
4-NOPD |
50 µg |
|
|
91 ± 10 |
|
|
|
|
MMS |
3 µL |
|
|
|
|
|
807 ± 34 |
|
|
|
|
|
|
|
|
|
|
With Activation |
DMSO |
|
|
21 ± 4B M |
24 ± 6 |
37 ± 6 |
137 ± 5 |
47 ± 8 |
Untreated |
|
|
30 ± 2B M |
28 ± 3 |
57 ± 5 |
164 ± 5 |
51 ± 6 |
|
Test item |
3 µg |
|
24 ± 1B M |
28 ± 3 |
44 ± 6 |
121 ± 11 |
53 ± 5 |
|
10 µg |
|
25 ± 5B M |
21 ± 2 |
40 ± 3 |
135 ± 10 |
48 ± 7 |
||
|
33 µg |
|
19 ± 2B M |
22 ± 5 |
39 ± 12 |
133 ± 25 |
50 ± 8 |
|
|
100 µg |
|
23 ± 3B M |
25 ± 6 |
40 ± 7 |
139 ± 9 |
45 ± 5 |
|
|
333 µg |
|
25 ± 5B M |
26 ± 6 |
45 ± 9 |
124 ± 10 |
45 ± 11 |
|
|
1000 µg |
|
15 ± 4B M P |
23 ± 5P |
34 ± 7P |
128 ± 24P |
52 ± 9P |
|
|
2500 µg |
|
18 ± 3B M P |
18 ± 5P M |
24 ± 8P M |
131 ± 25P M |
37 ± 6P M |
|
|
5000 µg |
|
15 ± 5P M B |
18 ± 3P M |
25 ± 7P M |
139 ± 16P M |
39 ± 6P M |
|
2-AA |
2.5 µg |
|
|
|
|
2959 ± 119 |
|
|
2-AA |
2.5 µg |
|
297 ± 15B M |
458 ± 9 |
|
|
|
|
2-AA |
10 µg |
|
|
|
|
|
666 ± 117 |
|
Congo red |
500 µg |
|
|
|
481 ± 56 |
|
|
|
|
|
|
|
|
|
|
|
|
Key to Positive Controls |
Key to Plate Postfix Codes |
||
|
|
||
NaN3 2-AA 4-NOPD MMS |
sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate |
P M B |
Precipitate Manual count Extensive bacterial growth |
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
No classification: No adverse effects observed
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