Registration Dossier
Registration Dossier
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EC number: 200-198-0 | CAS number: 54-21-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames assay:
The test chemical tested negative for mutagenicity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system.
In vitro mammalian chromosome aberration study:
The test chemical is not expected to produce mutachromosomal effects in Chinese hamster lung-derived fibroblasts or Chinese hamster ovary cells.
In vitro gene mutation study in mammalian cells
The test chemical tested negative for mutagenicity in CHO cells in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity of the chemical in CHO in the presence of metabolic activation due to invalid positive control data.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 17-04-2018 - 10-05-2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from study report.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- This study was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Details on mammalian cell type (if applicable):
- Not applicable
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254 induced S9 metabolic activation system
- Test concentrations with justification for top dose:
- 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was soluble in RO water - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- RO water
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- methylmethanesulfonate
- other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)
DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data
SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data
NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable
NUMBER OF CELLS EVALUATED: No data
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data
- OTHER: No data - Rationale for test conditions:
- No data
- 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 TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/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 control and vehicle control such an increase is not considered biologically relevant. - Statistics:
- No data
- Species / strain:
- S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- not specified
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data
RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100, cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate (T7 to T8), moderate inhibition was observed in the treated concentrations 0.501 mg/plate (T6) and there was no reduction in colony count as well as background lawn in any of the following concentrations tested; 0.002, 0.005, 0.016, 0.050 and 0.158 ( T1 to T5) mg/plate both in absence and in the presence of metabolic activation, when compared to that of the negative control group. Based on the results of pre-experiment following doses were selected for the main study trials: 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data - Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
- Executive summary:
Ames assay was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the given test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from experimental study report.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Principles of method if other than guideline:
- The purpose of this study was to assess toxic and genotoxic effects of test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation test using the Hprt and xprt genes
- Target gene:
- Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.
This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.
Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days. - Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Cell line used: Chinese Hamster Ovary (CHO) cells
- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable - Additional strain / cell type characteristics:
- other: Hypodiploid, modal No. 20
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
- Test concentrations with justification for top dose:
- 0, 0.0625, 0.125, 0.25 or 0.5 mM
- Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle: Sodium salicylate was easily dissolved in PBS. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- Phosphate-buffered saline
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: In medium with pre-incubation
DURATION
Pre-incubation:One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
Exposure duration:3 hours
Expression time:7 days
Selection time:14 days
Fixation time:7 days (harvest of cells)
SELECTION AGENT (mutation assays):6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays):Not applicable
STAIN (for cytogenetic assays):Crystal violet
NUMBER OF REPLICATIONS:A minimum of 2 replicates per dose concentration including negative and positive control.
NUMBER OF CELLS EVALUATED:5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.
DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding. - Rationale for test conditions:
- No data
- Evaluation criteria:
- The cell line was observed for gene mutation
- Statistics:
- No data
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- other: No data
- Cytotoxicity / choice of top concentrations:
- other: No data
- Vehicle controls validity:
- valid
- Positive controls validity:
- not valid
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No conclusions could be drawn regarding genotoxicity or cytotoxicity of the test chemical in CHO cells in the presence of metabolic activation due to invalid positive control data.
- Remarks on result:
- other: No mutagenic potential was observed in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity in the presence of metabolic activation due to invalid positive control data.
- Conclusions:
- The test chemical tested negative for mutagenicity in CHO cells in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity of the chemical in CHO in the presence of metabolic activation due to invalid positive control data.
- Executive summary:
An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, chemical was administered to CHO cells for 3 hrs at the dose levels of 0.0625, 0.125, 0.25 or 0.5 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. N-ethyl-N-nitrosourea (ENU)and7,12-dimethylbenz(a) anthraceneserved as positive.The positive control used in absence of metabolic activation (i.e.N-ethyl-N-nitrosourea) produced a significant increase in the number of revertant colonies whereas the positive control used in presence of metabolic activation (i.e. 7,12 -dimethylbenz(a)anthracene) did not. Without metabolic activation, the test chemical tested negative for mutagenicity in CHO cells. No conclusions could be reached regarding the mutagenicity of the test chemical in CHO cells in the presence of the metabolic activation system.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Remarks:
- Experimental data from various test chemicals
- Justification for type of information:
- Data for the target chemical is summarized based on the various test chemicals.
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Qualifier:
- according to guideline
- Guideline:
- other: As mentioned below
- Principles of method if other than guideline:
- WoE for the target CAS is summarized based on data from various test chemicals.
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- No data
- Species / strain / cell type:
- other: Chinese hamster lung-derived fibroblasts (CHL)
- Remarks:
- 7
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells:
The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970),
For cell lines:
- Absence of Mycoplasma contamination:
No data
- Number of passages if applicable:
4-day passages
- Methods for maintenance in cell culture:
The cell line was maintained in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Cell cycle length, doubling time or proliferation index :
The doubling time was approximately 15 hr.
- Modal number of chromosomes:
The modal chromosome number is 25 - Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Remarks:
- 8
- Details on mammalian cell type (if applicable):
- CHO cells were grown in MEM supplemented with 10% fetal calf serum, antibiotics and sodium bicarbonate. Stock cultures were maintained in culture flasks at 37 degree Celsius in a water-saturated CO2 incubator.
- Additional strain / cell type characteristics:
- not specified
- Cytokinesis block (if used):
- 7: 0.2 ug/ml colcemid was added to the culture 2 hr before cell harvesting
8: 0.1 ml of colchicine (0.01% in 2.5% Eagle's Minimal Essential Medium) was added at 16 hours post-exposure and left for 4 hours. - Metabolic activation:
- with and without
- Metabolic activation system:
- 7. no metabolic activation systems were applied
8. Aroclor-induced rat liver microsomal S9 cell fraction - Test concentrations with justification for top dose:
- 7. 0, 0.25 mg/ml. The top dose was expected to produce a 50% inhibition on cell growth, based on data from a pre-experiment.
8. 0 and 25 mg/ml. The top dose was half the dose that induced mitotic inhibition which was defined as one metaphase or less in 6000 CHO cells. - Vehicle / solvent:
- 7. DMSO
8. 2.5% Eagle's Minimal Essentil Medium (MEM supplemented with 2.5% fetal calf serum). - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- not specified
- Positive controls:
- not specified
- Positive control substance:
- not specified
- Remarks:
- 7
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 2.5% Eagle's Minimal Essentil Medium (MEM supplemented with 2.5% fetal calf serum).
- True negative controls:
- not specified
- Positive controls:
- other:
- Remarks:
- No positive controls were used, but several agents were tested for clastogenic effects in the study.
- Remarks:
- 8
- Details on test system and experimental conditions:
- 7. DURATION
- Exposure duration: 24 & 48 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: 50% cell growth inhibition
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
8. About 140,000 CHO cells were seeded on plastic dishes and kept in MEM with 10% fetal calf serum at 37°C for two to three days. Experiments were initiated when cells were confluent by 40-60%. The medium was removed from the dishes and replaced with test solutions. After 3 hours of exposure, the medium was removed, and the samples were washed with MEM, and fresh MEM was added to the dishes. Next, 0.1 ml of colchicine (0.01% in 2.5% Eagle's Minimal Essential Medium) was added at 16 hours post-exposure and left for 4 hours. Cells were then treated with 1% sodium citrate solution for 20 minutes and then immediately fixe in ethanol/acetic acid for 20 minutes. Sliders were next air-dried and stained in 2% orcein in 50% acetic acid/water, dehydrated, and mounted. 200 metaphases per sample were scored for chromosomal aberrations. The results were presented as the frequency of metaphases with chromosomal aberrations, the number of chromatid breaks per cell, and the number of chromatid exchanges per cell. - Evaluation criteria:
- 7. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%
8. Not specified - Statistics:
- 7. No data
8. Not specified - Species / strain:
- other: Chinese hamster lung-derived fibroblasts (CHL)
- Remarks:
- 7
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other:
- Remarks:
- Top dose was expected to produced a 50% inhibition on cell growth based on data from a pre-experiment.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- not specified
- Species / strain:
- Chinese hamster Ovary (CHO)
- Remarks:
- 8
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other:
- Remarks:
- Top dose was half the dose that produced 100% cytotoxicity.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- other:
- Remarks:
- A number of chemicals tested positive without and/or with metabolic activation
- Remarks on result:
- other: No mutagenic potential
- Conclusions:
- The test chemical is not expected to produce mutachromosomal effects in Chinese hamster lung-derived fibroblasts or Chinese hamster ovary cells.
- Executive summary:
The test chemical is not expected to produce mutachromosomal effects in Chinese hamster lung-derived fibroblasts or Chinese hamster ovary cells based on the data below.
Study 1
In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.25 mg/ml for 24 and 48 hr. DMSO was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).
Study 2
The test chemical was tested for clastogenic effects in Chinese hamster ovary (CHO) cells at 0 (solvent control) and 25 mg/ml, with and without metabolic activation (S9). The top dose was half the dose that induced mitotic inhibition which was defined as one metaphase or less in 6000 CHO cells. 200 metaphases per sample were scored for chromosomal aberrations. The results were presented as the frequency of metaphases with chromosomal aberrations, the number of chromatid breaks per cell, and the number of chromatid exchanges per cell. No significant effect was observed following treatment with the test chemical compared to solvent control data. No positive controls were used, however, several other agents that were evaluated in the study tested clearly positive for clastogenic effects without and/or with metabolic activation. This is taken as an indication that the assay was valid for the detection of clastogenic effects in CHO cells.
Referenceopen allclose all
TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT
Dose (mg/plate) |
R |
Without metabolic activation (-S9) |
With metabolic activation (+S9) |
||
TA100 |
TA 98 |
TA100 |
TA 98 |
||
NC (0.00) |
R1 |
122 |
24 |
126 |
25 |
R2 |
118 |
20 |
119 |
20 |
|
R3 |
124 |
19 |
121 |
22 |
|
T1 (0.002) |
R1 |
106 |
17 |
108 |
19 |
R2 |
110 |
19 |
112 |
17 |
|
R3 |
111 |
17 |
106 |
17 |
|
T2 (0.005) |
R1 |
108 |
19 |
112 |
21 |
R2 |
102 |
20 |
108 |
23 |
|
R3 |
104 |
18 |
110 |
20 |
|
T3 (0.016) |
R1 |
114 |
20 |
108 |
18 |
R2 |
106 |
22 |
110 |
16 |
|
R3 |
102 |
19 |
114 |
18 |
|
T4 (0.050) |
R1 |
116 |
21 |
106 |
21 |
R2 |
112 |
17 |
114 |
23 |
|
R3 |
118 |
18 |
108 |
19 |
|
T5 (0.158) |
R1 |
102 |
18 |
100 |
17 |
R2 |
98 |
17 |
96 |
18 |
|
R3 |
100 |
15 |
104 |
17 |
|
T6 (0.501) |
R1 |
36 (+ + +) |
2 (+ + +) |
42 ( + + + ) |
4 (+ + +) |
R2 |
26 (+ + +) |
5 (+ + +) |
34 (+ + +) |
3 (+ + +) |
|
R3 |
24 (+ + +) |
3 (+ + +) |
30 ( + + +) |
3 (+ + +) |
|
T7 (1.582) |
R1 |
0 (+) |
0 (+) |
0 (+) |
0 (+) |
R2 |
0 (+) |
0 (+) |
0 (+) |
0 (+) |
|
R3 |
0 (+) |
0 (+) |
0 (+) |
0 (+) |
|
T8 (5) |
R1 |
0 (+) |
0 (+) |
0 (+) |
0 (+) |
R2 |
0 (+) |
0 (+) |
0 (+) |
0 (+) |
|
R3 |
0 (+) |
0 (+) |
0 (+) |
0 (+) |
|
PC |
R1 |
1088 |
960 |
1584 |
1242 |
R2 |
1136 |
992 |
1616 |
1180 |
|
R3 |
1168 |
1008 |
1600 |
1208 |
|
NC = Negative control
PC = Positive control
R = Replicate
T = Test concentration (T8: Highest, T1: Lowest)
4-Nitro-o-phenylenediamine [10μg/plate]: TA 98
Sodium azide [10μg/plate]: TA 100,
2-Aminoanthracene [2.5μg/plate]: TA98, TA100
TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)
Dose (mg/plate) |
R |
In the Presence of Metabolic Activation (+S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
7 |
16 |
25 |
126 |
280 |
R2 |
6 |
15 |
20 |
119 |
272 |
|
R3 |
8 |
14 |
22 |
121 |
260 |
|
T1 (0.002) |
R1 |
4 |
13 |
19 |
108 |
242 |
R2 |
5 |
11 |
17 |
112 |
238 |
|
R3 |
5 |
10 |
17 |
106 |
232 |
|
T2 (0.005) |
R1 |
5 |
12 |
21 |
112 |
240 |
R2 |
4 |
13 |
23 |
108 |
248 |
|
R3 |
4 |
13 |
20 |
110 |
252 |
|
T3 (0.016) |
R1 |
6 |
14 |
18 |
108 |
256 |
R2 |
5 |
12 |
16 |
110 |
248 |
|
R3 |
4 |
13 |
18 |
114 |
264 |
|
T4 ((0.050) |
R1 |
6 |
15 |
21 |
106 |
260 |
R2 |
5 |
12 |
23 |
114 |
254 |
|
R3 |
5 |
14 |
19 |
108 |
268 |
|
T5 (0.158) |
R1 |
6 |
14 |
17 |
100 |
270 |
R2 |
6 |
15 |
18 |
96 |
266 |
|
R3 |
5 |
14 |
17 |
104 |
258 |
|
PC |
R1 |
168 |
480 |
1242 |
1584 |
1384 |
R2 |
186 |
452 |
1180 |
1616 |
1336 |
|
R3 |
170 |
492 |
1208 |
1600 |
1312 |
|
Dose (mg/plate) |
R |
In the Absence of Metabolic Activation (-S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
7 |
16 |
24 |
122 |
276 |
R2 |
6 |
14 |
20 |
118 |
264 |
|
R3 |
7 |
13 |
19 |
124 |
258 |
|
T1 (0.002) |
R1 |
5 |
12 |
17 |
106 |
232 |
R2 |
4 |
14 |
19 |
110 |
240 |
|
R3 |
5 |
11 |
17 |
111 |
236 |
|
T2 (0.005) |
R1 |
6 |
13 |
19 |
108 |
242 |
R2 |
4 |
15 |
20 |
102 |
238 |
|
R3 |
5 |
13 |
18 |
104 |
246 |
|
T3 (0.016) |
R1 |
5 |
14 |
20 |
114 |
240 |
R2 |
5 |
14 |
22 |
106 |
252 |
|
R3 |
6 |
15 |
19 |
102 |
236 |
|
T4 ((0.050) |
R1 |
6 |
14 |
21 |
116 |
254 |
R2 |
5 |
12 |
17 |
112 |
250 |
|
R3 |
6 |
13 |
18 |
118 |
262 |
|
T5 (0.158) |
R1 |
6 |
15 |
18 |
102 |
256 |
R2 |
6 |
15 |
17 |
98 |
260 |
|
R3 |
6 |
14 |
15 |
100 |
252 |
|
PC |
R1 |
180 |
1320 |
960 |
1088 |
1824 |
R2 |
174 |
1272 |
992 |
1136 |
1840 |
|
R3 |
170 |
1344 |
1008 |
1168 |
1888 |
|
NC= Negative Control,T=Test concentration (T5: Highest, T1: Lowest),R= Replicate
PC= Positive
control 2-Aminoanthracene
[2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100
2- Aminoanthracene [10μg/plate]:TA
102 Sodium azide [10μg/plate]: TA
1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate] Methyl methanesulfonate [4μl/plate]: TA 102
TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)
Dose (mg/plate) |
R |
In the Presence of Metabolic Activation (+S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
8 |
16 |
28 |
128 |
272 |
R2 |
6 |
14 |
25 |
123 |
258 |
|
R3 |
7 |
13 |
23 |
124 |
260 |
|
T1 (0.002) |
R1 |
5 |
11 |
20 |
120 |
230 |
R2 |
4 |
10 |
20 |
118 |
238 |
|
R3 |
5 |
13 |
19 |
121 |
244 |
|
T2 (0.005) |
R1 |
5 |
13 |
21 |
123 |
250 |
R2 |
4 |
12 |
23 |
120 |
246 |
|
R3 |
4 |
12 |
24 |
124 |
240 |
|
T3 (0.016) |
R1 |
6 |
10 |
19 |
122 |
238 |
R2 |
5 |
14 |
25 |
119 |
246 |
|
R3 |
5 |
15 |
26 |
125 |
254 |
|
T4 ((0.050) |
R1 |
6 |
13 |
25 |
123 |
248 |
R2 |
6 |
12 |
24 |
124 |
256 |
|
R3 |
5 |
15 |
23 |
124 |
250 |
|
T5 (0.158) |
R1 |
7 |
15 |
26 |
125 |
258 |
R2 |
6 |
14 |
22 |
123 |
264 |
|
R3 |
6 |
14 |
25 |
123 |
260 |
|
PC |
R1 |
162 |
380 |
1344 |
1440 |
1680 |
R2 |
174 |
440 |
1360 |
1472 |
1704 |
|
R3 |
180 |
420 |
1384 |
1504 |
1712 |
|
Dose (mg/plate) |
R |
In the Absence of Metabolic Activation (-S9) |
||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||
NC (0.00) |
R1 |
7 |
15 |
26 |
126 |
260 |
R2 |
6 |
13 |
23 |
123 |
252 |
|
R3 |
7 |
13 |
22 |
120 |
248 |
|
T1 (0.002) |
R1 |
4 |
10 |
21 |
108 |
232 |
R2 |
5 |
13 |
23 |
102 |
228 |
|
R3 |
5 |
11 |
19 |
104 |
236 |
|
T2 (0.005) |
R1 |
4 |
14 |
18 |
106 |
234 |
R2 |
4 |
11 |
20 |
112 |
230 |
|
R3 |
4 |
12 |
23 |
110 |
242 |
|
T3 (0.016) |
R1 |
5 |
13 |
24 |
114 |
248 |
R2 |
6 |
14 |
22 |
108 |
240 |
|
R3 |
4 |
12 |
19 |
111 |
252 |
|
T4 ((0.050) |
R1 |
6 |
13 |
23 |
114 |
250 |
R2 |
5 |
13 |
24 |
116 |
254 |
|
R3 |
5 |
14 |
20 |
120 |
246 |
|
T5 (0.158) |
R1 |
6 |
14 |
24 |
123 |
258 |
R2 |
6 |
14 |
23 |
120 |
248 |
|
R3 |
5 |
13 |
25 |
121 |
250 |
|
PC |
R1 |
180 |
1168 |
890 |
1248 |
1552 |
R2 |
178 |
1184 |
924 |
1280 |
1520 |
|
R3 |
182 |
1216 |
916 |
1304 |
1568 |
|
NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest), R= Replicate
PC= Positive
control 2-Aminoanthracene
[2.5μg/plate]: TA 1537, TA1535, TA98, TA100
2-Aminoanthracene [10μg/plate]:TA
102 Sodium azide
[10μg/plate]: TA 1535, TA
100,
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate] Methyl methanesulfonate [4μl/plate]: TA 102
TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)
Dose (mg/plate) |
In the presence of Metabolic Activation (+S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
7.00 |
1.00 |
15.00 |
1.00 |
22.33 |
2.52 |
122.00 |
3.61 |
270.67 |
10.07 |
T1 (0.002) |
4.67 |
0.58 |
11.33 |
1.53 |
17.67 |
1.15 |
108.67 |
3.06 |
237.33 |
5.03 |
T2 (0.005) |
4.33 |
0.58 |
12.67 |
0.58 |
21.33 |
1.53 |
110.00 |
2.00 |
246.67 |
6.11 |
T3 (0.016) |
5.00 |
1.00 |
13.00 |
1.00 |
17.33 |
1.15 |
110.67 |
3.06 |
256.00 |
7.02 |
T4 (0.050) |
5.33 |
0.58 |
13.67 |
1.53 |
21.00 |
2.00 |
109.33 |
4.16 |
260.67 |
8.00 |
T5 (0.158) |
5.67 |
0.58 |
14.33 |
0.58 |
17.33 |
0.58 |
100.00 |
4.00 |
264.67 |
6.11 |
PC |
174.67 |
9.87 |
474.67 |
20.53 |
1210.00 |
31.05 |
1600.00 |
16.00 |
1344.00 |
36.66 |
Dose (mg/plate) |
In the Absence of Metabolic Activation (-S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
6.67 |
0.58 |
14.33 |
1.53 |
21.00 |
2.65 |
121.33 |
3.06 |
266.00 |
9.17 |
T1 (0.002) |
4.67 |
0.58 |
12.33 |
1.53 |
17.67 |
1.15 |
109.00 |
2.65 |
236.00 |
4.00 |
T2 (0.005) |
5.00 |
1.00 |
13.67 |
1.15 |
19.00 |
1.00 |
104.67 |
3.06 |
242.00 |
4.00 |
T3 (0.016) |
5.33 |
0.58 |
14.33 |
0.58 |
20.33 |
1.53 |
107.33 |
6.11 |
242.67 |
8.33 |
T4 (0.050) |
5.67 |
0.58 |
13.00 |
1.00 |
18.67 |
2.08 |
115.33 |
3.06 |
255.33 |
6.11 |
T5 (0.158) |
6.00 |
0.00 |
14.67 |
0.58 |
16.67 |
1.53 |
100.00 |
2.00 |
256.00 |
4.00 |
PC |
174.67 |
5.03 |
1312.00 |
36.66 |
986.67 |
24.44 |
1130.67 |
40.27 |
1850.67 |
33.31 |
NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100 Methyl methanesulfonate [4μl/plate]: TA 102
2-Aminoanthracene [10μg/plate]:TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]
TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)
Dose (mg/plate) |
In the presence of Metabolic Activation (+S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
7.00 |
1.00 |
14.33 |
1.53 |
25.33 |
2.52 |
125.00 |
2.65 |
263.33 |
7.57 |
T1 (0.002) |
4.67 |
0.58 |
11.33 |
1.53 |
19.67 |
0.58 |
119.67 |
1.53 |
237.33 |
7.02 |
T2 (0.005) |
4.33 |
0.58 |
12.33 |
0.58 |
22.67 |
1.53 |
122.33 |
2.08 |
245.33 |
5.03 |
T3 (0.016) |
5.33 |
0.58 |
13.00 |
2.65 |
23.33 |
3.79 |
122.00 |
3.00 |
246.00 |
8.00 |
T4 (0.050) |
5.67 |
0.58 |
13.33 |
1.53 |
24.00 |
1.00 |
123.67 |
0.58 |
251.33 |
4.16 |
T5 (0.158) |
6.33 |
0.58 |
14.33 |
0.58 |
24.33 |
2.08 |
123.67 |
1.15 |
260.67 |
3.06 |
PC |
172.00 |
9.17 |
413.33 |
30.55 |
1362.67 |
20.13 |
1472.00 |
32.00 |
1698.67 |
16.65 |
Dose (mg/plate) |
In the Absence of Metabolic Activation (-S9) |
|||||||||
TA 1537 |
TA 1535 |
TA 98 |
TA 100 |
TA 102 |
||||||
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
MEAN |
SD |
|
NC (0.00) |
6.67 |
0.58 |
13.67 |
1.15 |
23.67 |
2.08 |
123.00 |
3.00 |
253.33 |
6.11 |
T1 (0.002) |
4.67 |
0.58 |
11.33 |
1.53 |
21.00 |
2.00 |
104.67 |
3.06 |
232.00 |
4.00 |
T2 (0.005) |
4.00 |
0.00 |
12.33 |
1.53 |
20.33 |
2.52 |
109.33 |
3.06 |
235.33 |
6.11 |
T3 (0.016) |
5.00 |
1.00 |
13.00 |
1.00 |
21.67 |
2.52 |
111.00 |
3.00 |
246.67 |
6.11 |
T4 (0.050) |
5.33 |
0.58 |
13.33 |
0.58 |
22.33 |
2.08 |
116.67 |
3.06 |
250.00 |
4.00 |
T5 (0.158) |
5.67 |
0.58 |
13.67 |
0.58 |
24.00 |
1.00 |
121.33 |
1.53 |
252.00 |
5.29 |
PC |
180.00 |
2.00 |
1189.33 |
24.44 |
910.00 |
17.78 |
1277.33 |
28.10 |
1546.67 |
24.44 |
NC= Negative Control, T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation
PC= Positive control
2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100
2-Aminoanthracene [10μg/plate]: TA 102
Sodium azide [10μg/plate]: TA 1535, TA 100
4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]
Methyl methanesulfonate: [4μl/plate]: TA 102
Table 1A.Effect of sodium salicylate exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).
|
With S9 |
Without S9 |
||
|
with TG |
without TG |
with TG |
without TG |
Neg. control |
0/4 |
184/4 |
0/4 |
208/4 |
Pos. control |
0/4 |
221/4 |
12/4 |
143/4 |
0.0625 mM |
2/4a |
218/4 |
0/4 |
198/4 |
0.125 mM |
0/4 |
226/4 |
0/4 |
185/4 |
0.25 mM |
0/4 |
177/4 |
0/4 |
181/4 |
0.5 mM |
0/4 |
185/4 |
0/4 |
198/4 |
a)2 very diffuse colonies were found in one single well.
Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to sodium salicylate in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.
|
With S9 |
Without S9 |
Neg. control |
N/A |
N/A |
Pos. control |
N/A |
3.08x10-4 |
0.0625 mM |
N/Aa |
N/A |
0.125 mM |
N/A |
N/A |
0.25 mM |
N/A |
N/A |
0.5 mM |
N/A |
N/A |
a)Since only diffuse colonies were found in one single well (see Table 1A), these diffuse colonies were not regarded as reliable and true colonies since the cells seemed to be apoptotic.
Study 8:
Table 1.Tabular data for solvent control, the test chemical, and for a selection of other agents that were tested for clastogenic effects in CHO cells.
Compound (conc. In mg/ml) |
Activation |
% metaphases with chromosome aberrations |
Chromatid breaks per cell |
Chromatid exchanges per cell |
Solvent control (0) |
With and without S9 |
0.7 |
0.01 |
0.00 |
Test chemical (10) |
-S9 |
1.5 |
0.02 |
0.00 |
|
+S9 |
1.0 |
0.01 |
0.00 |
4-Methylcatechol (0.01) |
-S9 |
20.0 |
0.10 |
0.73 |
|
+S9 |
0.0 |
0.00 |
0.00 |
Pyrogallol (0.1) |
-S9 |
22.6 |
0.14 |
1.08 |
|
+S9 |
18.0 |
0.06 |
0.84 |
Eugenol (0.05) |
-S9 |
0.5 |
0.01 |
0.00 |
|
+S9 |
13.9 |
0.17 |
0.24 |
Note: Solvent control data was presented only as mean values from the two experiments combined (with and without metabolic activation).
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Study 1
The test chemical was tested for clastogenic effects in mice. In the sister chromatid exchange (SCE) assay, the test chemical was given to mice in two separate trials. In trial 1, the chemical was given by oral gavage at 0 and 350 mg/kg bw (single dose). In trial 2, the test chemical was given by intraperitoneal injection at 0, 25, 50 and 100 mg/kg bw (single dose). In the chromosomal aberration assay, the test chemical was given to mice in two separate trials. In trial 1, the chemical was given to mice by oral gavage at 0 and 350 mg/kg bw (single dose). In trial 2, the test chemical was given by intraperitoneal injection at 0, 50, 100 and 200 mg/kg bw (single dose). Dose levels were selected based on the oral LD50dose for acetyl salicylic acid in mice, i.e. the oral dose was 1/3 of the oral LD50dose and the highest intraperitoneal injection dose was either 1/10 (SCE assay) or 1/5 (aberration assay) of the oral LD50dose. For all trials, the bone marrows were expelled at 24 hours after treatment to detect either SCE or chromosome aberrations (gaps excluded). The test chemical failed to produce any significant increase in the number of SCE per cell under the experimental conditions. In the chromosomal aberration assay, a weak but statistically significant positive response was observed following intraperitoneal injection at 200 mg/kg (mean, 3.5% aberrant cells at 200 mg/kg vs. mean, 1.75% at 0 mg/kg). Likewise, a weak but statistically significant positive response was observed following oral gavage treatment at 350 mg/kg (mean, 4.20% at 350 mg/kg vs. mean, 2.20% at 0 mg/kg). Treatment with the positive control cyclophosphamide at 25 mg/kg (i.p. injection) resulted in a chromosomal aberration frequency of 12.20%. The weak positive responses in the chromosome aberration assays were attributed to the ablitiy of the test chemical to biotransform to salicylic acid which per se showed no evidence of genototoxicity in the assay.
Study 2
The chemical was given via diet at 0 and 10 mM for 3 days to 1-2 day old Berlin K males. A single dose of 10 mM corresponded to the LD50 value and was regarded to be the maximally tolerated dose in the insects. Treated males were mated individually with 3 Basc virgin females. A sequence of three brood periods (each lasting 3 days) was then initiated. At the end of each 3-day breeding period, treated males were transferred to new vials and mated individually with 3 virgin females. Typically, at least 1000 F1 females were handled in each brood. Sex-linked recessive lethal mutations were scored in the F2 and F3 generations. Treatment with the test chemical resulted in no significant increase in the frequency of sex-linked recessive lethal mutation in any of the broods or in all broods combined when compared to concurrent negative control data. Treatment with 1,2 dichloroethane, which is a probable human carcinogen, at 50 mM resulted in a significant increase in the frequency of sex-linked recessive lethal mutations in brood 2 and in all broods combined when compared to control data, indicating that the assay was valid for detecting mutagenic effects.
Link to relevant study records
- Endpoint:
- genetic toxicity in vivo, other
- Remarks:
- In vivo sister-chromatid exchange assay
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from peer reviewed publication.
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- In vivo sister chromatid exchange by the oral route was performed to determine the mutagenic nature of the given test chemical.
- GLP compliance:
- not specified
- Type of assay:
- sister chromatid exchange assay
- Species:
- mouse
- Strain:
- Swiss
- Remarks:
- albino
- Details on species / strain selection:
- No data
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Division of Laboratory animals, Central Drug Research Institute, Lucknow
- Age at study initiation: 10-12 week old
- Weight at study initiation: 30 g
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: They were kept five per
cage with husk bedding
- Diet (e.g. ad libitum): Standard rodent
pellet diet (Gold Mohor, Lipton India Ltd., Chandigarb, India) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 ± 2°C
- Humidity (%): 60_+ 5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark
IN-LIFE DATES: From: To: No data - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: distilled water in 2% gum acacia
- Justification for choice of solvent/vehicle: The test chemical was soluble in distilled water in 2% gum acacia
- Concentration of test material in vehicle: 0 or 350 mg/Kg
- Amount of vehicle (if gavage or dermal): 0.3mL/mouse
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data - Details on exposure:
- Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the oral dose was 1/3 of the oral LD50 dose.
- Duration of treatment / exposure:
- Single dose
- Frequency of treatment:
- Single dose
- Post exposure period:
- The test chemicals were administered as a single i.p. injection 1 h after tablet implantation. Colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled
- Remarks:
- 0 or 350 mg/Kg
- No. of animals per sex per dose:
- Total: 15 male mice
0 mg/Kg: 5 male mice
350 mg/Kg: 5 male mice - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Not used.
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. In the single-dose oral study, the test chemical was gavaged with distilled water in 2% gum acacia (0.3 ml/mouse) at the dose of 350 mg/kg half an hour after tablet implantation to different groups of 5 animals each. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation.
DETAILS OF SLIDE PREPARATION: Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique. All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested.
METHOD OF ANALYSIS: Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first (M 1), second (M 2) and third (M 3) division metaphases
OTHER: No data - Evaluation criteria:
- The bone marrow cells were observed for sister chromatid exchanges
- Statistics:
- Student's t-test was used to compare the results of the treated series with the respective controls for SCE, MI and RI in the oral study conducted. Level of statistical significance was set at p < 0.05.
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Remarks on result:
- other: No mutagenic potential
- Additional information on results:
- No data
- Conclusions:
- The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice treated at 350 mg/kg bw (single dose) when compared to concurrent negative control data.
- Executive summary:
In vivo sister chromatid exchange was performed to determine the mutagenic nature of the given test chemical. The study was performed using Swiss albino male mice. The test chemical was dissolved in 2% gum acacia in distilled water at dose level of 0 or 350 mg/Kg. Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. In the single-dose oral study, test chemical was gavaged with distilled water in 2% gum acacia (0.3 ml/mouse) at the dose of 350 mg/kg half an hour after tablet implantation to different groups of 5 animals each. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique. All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first (M 1), second (M 2) and third (M 3) division metaphases. The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice treated at 350 mg/kg bw (single dose) when compared to concurrent negative control data.
- Endpoint:
- genetic toxicity in vivo, other
- Remarks:
- In vivo sister chromatid exchange assay
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from peer reviewed publication.
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- In vivo sister chromatid exchange by the intraperitoneal route was performed to determine the mutagenic nature of the given test chemical.
- GLP compliance:
- not specified
- Type of assay:
- sister chromatid exchange assay
- Species:
- mouse
- Strain:
- Swiss
- Remarks:
- albino
- Details on species / strain selection:
- No data
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Division of Laboratory animals, Central Drug Research Institute, Lucknow
- Age at study initiation: 10-12 week old
- Weight at study initiation: 30 g
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: They were kept five per
cage with husk bedding
- Diet (e.g. ad libitum): Standard rodent
pellet diet (Gold Mohor, Lipton India Ltd., Chandigarb, India) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 ± 2°C
- Humidity (%): 60_+ 5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark
IN-LIFE DATES: From: To: No data - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
- Concentration of test material in vehicle: 0, 25, 50 or 100 mg/Kg
- Amount of vehicle (if gavage or dermal): 0.3mL/mouse
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data - Details on exposure:
- Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the highest intraperitoneal injection dose was 1/10 of the oral LD50 dose.
- Duration of treatment / exposure:
- Single dose
- Frequency of treatment:
- Single dose
- Post exposure period:
- The test chemicals were administered as a single i.p. injection 1 h after tablet implantation. Colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled
- Remarks:
- 0, 25, 50 or 100 mg/Kg
- No. of animals per sex per dose:
- Total: 25 male mice
0 mg/Kg: 5 male mice
25 mg/Kg: 5 male mice
50 mg/Kg: 5 male mice
100 mg/Kg: 5 male mice
Positive control: 5 male mice - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Mitomycin C
- Justification for choice of positive control(s): No data
- Route of administration: Intraperitoneal
- Doses / concentrations: 1.5 mg/Kg - Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. The test chemicals were administered as a single i.p. injection 1 h after tablet implantation. Three doses (25, 50 and 100 mg/kg) of the test chemical were injected i.p. in DMSO (75 µl/mouse) to different groups of 5 animals each. Negative control mice were injected with 75 µl DMSO while mitomycin C was used as a positive control at a dose of 1.5 mg/kg of body weight
DETAILS OF SLIDE PREPARATION: For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique. All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested.
METHOD OF ANALYSIS: Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first (M 1), second (M 2) and third (M 3) division metaphases
OTHER: No data - Evaluation criteria:
- The bone marrow cells were observed for sister chromatid exchanges
- Statistics:
- Student's t-test was used to compare the results of the treated series with the respective controls for SCE, MI and RI in the i.p. study conducted. Level of statistical significance was set at p < 0.05.
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: No mutagenic potential
- Additional information on results:
- No data
- Conclusions:
- The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice i.p. treated at up to 100 mg/kg bw (single dose) when compared to concurrent negative control data. Treatment with the positive control produced expected increases in the number of SCE per cells, thus confirming the validity of the assay.
- Executive summary:
In vivo sister chromatid exchange was performed to determine the mutagenic nature of the given test chemical. The study was performed using Swiss albino male mice. The test chemical was dissolved in DMSO at dose level of 0, 25, 50 or 100 mg/Kg. Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. The test chemical was administered as a single i.p. injection 1 h after tablet implantation. Three doses (25, 50 and 100 mg/kg) of test chemical were injected i.p. in DMSO (75µl/mouse) to different groups of 5 animals each. Negative control mice were injected with 75µl DMSO while mitomycin C was used as a positive control at a dose of 1.5 mg/kg of body weight. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique. All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice i.p. treated at up to 100 mg/kg bw (single dose) when compared to concurrent negative control data. Treatment with the positive control produced expected increases in the number of SCE per cells, thus confirming the validity of the assay.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from peer reviewed publication
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- In vitro chromosome aberration study by the intraperitoneal route was performed to determine the mutagenic nature of the given test chemical.
- GLP compliance:
- not specified
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- mouse
- Strain:
- Swiss
- Remarks:
- albino
- Details on species / strain selection:
- No data
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Division of Laboratory animals, Central Drug Research Institute, Lucknow
- Age at study initiation: 10-12 week old
- Weight at study initiation: 30 g
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: They were kept five per
cage with husk bedding
- Diet (e.g. ad libitum): Standard rodent
pellet diet (Gold Mohor, Lipton India Ltd., Chandigarb, India) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 ± 2°C
- Humidity (%): 60_+ 5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark
IN-LIFE DATES: From: To: No data - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
- Concentration of test material in vehicle: 0, 50, 100 or 200 mg/Kg
- Amount of vehicle (if gavage or dermal): 0.3mL/mouse
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data - Details on exposure:
- Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the highest intraperitoneal injection dose was 1/5 of the oral LD50 dose.
- Duration of treatment / exposure:
- Single dose
- Frequency of treatment:
- Single dose
- Post exposure period:
- After 22 hours of treatment, the animals were injected with colchicine (2 mg/kg) and 2 hours later they were sacrificed by cervical dislocation.
- Remarks:
- 0, 50, 100 or 200 mg/Kg
- No. of animals per sex per dose:
- Total: 21 male mice
0 mg/Kg: 4 male mice
50 mg/Kg: 4 male mice
100 mg/Kg: 4 male mice
200 mg/Kg: 4 male mice
Positive control: 5 male mice - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide at 25 mg/kg bw
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- Bone marrow chromosomes were prepared and slides were stained with Giemsa. All the slides were coded and 100 well spread metaphase cells were scored per animal.
- Evaluation criteria:
- Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. Chromosomal aberrations were scored following the method of WHO and previously published work. The aberrations frequencies of chromatid and chromosome types per cell were calculated. Gaps were recorded but not included in the frequency of aberrations per cell.
- Statistics:
- Statistical calculations were carried out from the percentages of aberrant cells. Student's t-test was used to compare the results of the treated series with the respective controls for chromosomal aberrations, mitotic indices and replicative indices.
- Sex:
- male
- Genotoxicity:
- positive
- Remarks:
- At top dose, a weak positive response was observed
- Toxicity:
- yes
- Remarks:
- At top dose, 52% degree of cytotoxicity was observed
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- No data
- Conclusions:
- Treatment with the test chemical at 200 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.
- Executive summary:
In vivo chromosome aberration study was performed to determine the mutagenic nature of the given test chemical. The study was performed using male Swiss albino mice. Three doses (50, 100 and 200 mg/kg) were dissolved in DMSO and injected i.p. (75 µl/mouse). Bone marrow chromosomes were prepared and slides were stained with Giemsa. All the slides were coded and 100 well spread metaphase cells were scored per animal. Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. Chromosomal aberrations were scored following previously published work. The aberrations frequencies of chromatid and chromosome types per cell were calculated. Gaps were recorded but not included in the frequency of aberrations per cell. Treatment with the test chemical at 200 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Justification for type of information:
- Data is from peer reviewed publication
- Qualifier:
- according to guideline
- Guideline:
- other: Refer below principle
- Principles of method if other than guideline:
- In vitro chromosome aberration study by the oral route was performed to determine the mutagenic nature of the test chemical
- GLP compliance:
- not specified
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- mouse
- Strain:
- Swiss
- Remarks:
- albino
- Details on species / strain selection:
- No data
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Division of Laboratory animals, Central Drug Research Institute, Lucknow
- Age at study initiation: 10-12 week old
- Weight at study initiation: 30 g
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: They were kept five per
cage with husk bedding
- Diet (e.g. ad libitum): Standard rodent
pellet diet (Gold Mohor, Lipton India Ltd., Chandigarb, India) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 ± 2°C
- Humidity (%): 60_+ 5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark
IN-LIFE DATES: From: To: No data - Route of administration:
- oral: gavage
- Vehicle:
- - Vehicle(s)/solvent(s) used: 2% gum acacia in distilled water
- Justification for choice of solvent/vehicle: The test chemical was soluble in distilled water in 2% gum acacia
- Concentration of test material in vehicle: 0 or 350 mg/Kg
- Amount of vehicle (if gavage or dermal): 0.3mL/mouse
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data - Details on exposure:
- Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the oral dose was 1/3 of the oral LD50 dose.
- Duration of treatment / exposure:
- Single dose
- Frequency of treatment:
- Single dose
- Post exposure period:
- After 22 hours of treatment, the animals were injected with colchicine (2 mg/kg) and 2 hours later they were sacrificed by cervical dislocation.
- Remarks:
- 0 or 350 mg/Kg
- No. of animals per sex per dose:
- 0 mg/Kg: 5 male mice
350 mg/Kg: 5 male mice - Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Not used.
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- Bone marrow chromosomes were prepared and slides were stained with Giemsa. All the slides were coded and 100 well spread metaphase cells were scored per animal.
- Evaluation criteria:
- Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. Chromosomal aberrations were scored following the method of WHO and previously published work. The aberrations frequencies of chromatid and chromosome types per cell were calculated. Gaps were recorded but not included in the frequency of aberrations per cell.
- Statistics:
- Statistical calculations were carried out from the percentages of aberrant cells. Student's t-test was used to compare the results of the treated series with the respective controls for chromosomal aberrations, mitotic indices and replicative indices.
- Sex:
- male
- Genotoxicity:
- positive
- Remarks:
- Weak positive response
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- No data
- Conclusions:
- Treatment with the test chemical at 350 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.
- Executive summary:
In vivo chromosome aberration study was performed to determine the mutagenic nature of the given test chemical. The study was performed using male Swiss albino mice. The test chemical was dissolved in 2% gum acacia in distilled water at a single oral dose of 0 or 350 mg/Kg. Negative control mice were gavaged only 2% gum acacia in distilled water. After 22 h of chemical treatment the animals were injected with colchicine (2 mg/kg) and 2 h later they were killed by cervical dislocation. Bone marrow chromosomes were prepared and slides were stained with Giemsa. All the slides were coded and 100 well spread metaphase cells were scored per animal. Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. Chromosomal aberrations were scored following the method of WHO and previously published work. The aberrations frequencies of chromatid and chromosome types per cell were calculated. Gaps were recorded but not included in the frequency of aberrations per cell. Treatment with the test chemical at 350 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.
- Endpoint:
- in vivo insect germ cell study: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- EPA OPPTS 870.5275 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)
- GLP compliance:
- not specified
- Type of assay:
- Drosophila SLRL assay
- Species:
- Drosophila melanogaster
- Strain:
- other: Berlin K
- Sex:
- male/female
- Route of administration:
- oral: feed
- Vehicle:
- The test chemical was dissolved in 5% sucrose solution containing 2% DMSO if necessary
- Details on exposure:
- The test chemical was given by diet to 1-2 day old Berlin K males for 3 consecutive days, normally at the single maximally tolerated dose level (up to the LD50).
- Duration of treatment / exposure:
- 3 days
- Frequency of treatment:
- daily by diet
- Post exposure period:
- After treatment, a sequence of three brood periods (each lasting 3 days) was then initiated. At the end of each 3-day breeding period, treated males were transferred to new vials and mated individually with 3 virgin females. Typically, at least 1000 F1 females were handled in each brood.
- Remarks:
- 0 (vehicle and 10 mM
- No. of animals per sex per dose:
- Number of males not specified but at least 1000 F1 females were handled in each brood.
- Control animals:
- yes, plain diet
- Positive control(s):
- 1,2 dichloroethane, which is a probable carcinogen in humans, was evaluated in addition to the test chemical for mutagenic effects in Drosophila.
- Tissues and cell types examined:
- X-chromosomes
- Details of tissue and slide preparation:
- No details given.
- Evaluation criteria:
- Recessive lethal mutations frequencies in brood I, II, III, and I to III, in F2 and F3 generations, following treatment with the test chemical in males, were evaluated against solven control data using statistical analysis.
- Statistics:
- Kastenbaum-Bowman test
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Three days treatment with the test chemical Drosophila at LD50 resulted in no significant increase in the frequency of sex-linked recessive lethal mutations in any of the broods or in all broods combined when compared to concurrent negative control data.
- Executive summary:
The chemical was given via diet at 0 and 10 mM for 3 days to 1-2 day old Berlin K males. A single dose of 10 mM corresponded to the LD50 value and was regarded to be the maximally tolerated dose in the insects. Treated males were mated individually with 3 Basc virgin females. A sequence of three brood periods (each lasting 3 days) was then initiated. At the end of each 3-day breeding period, treated males were transferred to new vials and mated individually with 3 virgin females. Typically, at least 1000 F1 females were handled in each brood. Sex-linked recessive lethal mutations were scored in the F2 and F3 generations. Treatment with the test chemical resulted in no significant increase in the frequency of sex-linked recessive lethal mutation in any of the broods or in all broods combined when compared to concurrent negative control data. Treatment with 1,2 dichloroethane, which is a probable human carcinogen, at 50 mM resulted in a significant increase in the frequency of sex-linked recessive lethal mutations in brood 2 and in all broods combined when compared to control data, indicating that the assay was valid for detecting mutagenic effects.
Referenceopen allclose all
Table: In vivo sister chromatid exchanges induced by test chemical in mice after oral administration
Treatment |
SCE/cell of 5 animals |
SCE/cell (mean ± SD)a |
Replicative indices mean ± SD)a |
Solvent control(Gum acacia) |
4.4, 3.9, 5.1, 4.6, 4.7 |
4.54±0.43 |
1.83±0.10 |
Test chemical (350 mg/kg) |
5.6, 5.9, 4.3, 5.1, 4.7 |
5.12±0.64 |
1.82±0.07 |
a) Mean ± SD of 5 animals (30 cells/animal). No significant effect was observed using Student's t-test.
Table: In vivo sister chromatid exchanges induced by test chemical in mice after intraperitoneal administration
Treatment |
SCE/cell of 5 animals |
SCE/cell (mean ± SD)a |
Replicative indices mean ± SD)a |
Solvent control(DMSO) |
4.5, 4.5, 4.0, 4.8, 5.5 |
4.66±0.55 |
1.86±0.09 |
Test chemical |
|
|
|
25 |
5.5, 4.4, 4.5, 5.9, 4.6 |
4.98±0.67 |
1.78±0.06 |
50 |
4.5, 4.2, 5.9, 5.5, 5.4 |
5.10±0.71 |
1.88±0.09 |
100 |
4.0, 4.5, 4.9, 5.5, 5.0 |
4.78±0.56 |
1.82± 0.10 |
Mitomycin C (1.5 mg/Kg ) |
18.5, 19.5, 23.2, 18.1, 16.6 |
19.18±2.47 |
1.77±0.18 |
a) Mean ± SD of 5 animals (30 cells/animal).
Table: In vivo chromosome aberration study induced by test chemical in mice after intaperitoneal administration
Treatment (mg/kg bw) |
Gapsa |
Aberrations/cellb |
Aberrant cells % (mean±SD)c |
Mitotic indices (mean±SD)c |
|
Chromatid type |
Chromosome type |
||||
Solvent control |
8
|
0.015
|
0.002
|
1.75±0.50
|
2.79±0.65 |
Test chemical |
|
|
|
|
|
50 |
8 |
0.020 |
0.000 |
2.00±0.82 |
3.54±0.75 |
100 |
7 |
0.027 |
0.000 |
2.75±1.26 |
3.95±0.91 |
250 |
8 |
0.027 |
0.0075 |
3.50±0.58** |
1.69±0.33 |
Cyclophosphamide at 25 mg/kg bw |
34 |
0.130 |
0.020 |
12.20±2.16 |
3.50±0.78 |
Salicylic acid |
|
|
|
|
|
50 |
9 |
0.0150 |
0.000 |
1.50 ± 0.58 |
4.11 ± 0.69 |
100 |
7 |
0.0175 |
0.000 |
1.75 ± 0.50 |
3.46 ± 0.96 |
200 |
11 |
0.0150 |
0.000 |
1.75 ± 0.96 |
3.20 ± 0.98 |
a Total chromatid and chromosome gaps at each dose were recorded but not included as aberration/cell.
b Total number of aberrations (chromatid or chromosome type)/total number of cells scored per group. Results are of 4 animals (100 cells/animal).
c Results at each dose wee compared to those of the control using Student's t-test, *p < 0.02, * * p < 0.01
Table: In vivo chromosome aberration study induced by test chemical in mice after oral administration
Treatment |
Gapsa |
Aberrations/cellb |
Aberrant cells % (mean±SD)c |
Mitotic indices (mean±SD)c |
|
Chromatid type |
Chromosome type |
||||
Solvent control |
9 |
0.022 |
0.00 |
2.20 ± 0.84 |
2.49 ± 0.55 |
Test chemical (350 mg/kg bw) |
18 |
0.034 |
0.008 |
4.20 ± 1.09** |
2.60 ± 0.37 |
Salicylic acid (350 mg/kg) |
12 |
0.018 |
0.008 |
2.60 ± 0.89 |
3.27 ± 0.48* |
a Total chromatid and chromosome gaps at each dose were recorded but not included as aberration/cell.
b Total number of aberrations (chromatid or chromosome type)/total number of cells scored per group. Results are of 5 animals (100 cells/animal).
c Results at each dose wee compared to those of the control using Student's t-test, *p < 0.05, * * p < 0.02, * * * p < 0.01.
Table 1. Data for solvent control and for the positive control (1,2-dichloroethane at 50 mM).
Conc. |
Brood |
Days after treatment |
Number of X-chromosomes tested |
Number of lethals |
Recessive lethal mutation frequency (%) |
P values |
0 |
I |
0-3 |
9565 |
24 |
0.25 |
NS |
0 |
II |
4-6 |
6500 |
4 |
0.06 |
NS |
0 |
III |
7-9 |
5983 |
19 |
0.32 |
NS |
0 |
I-III |
0-9 |
22048 |
47 |
0.21 |
NS |
50 |
I |
0-3 |
1185 |
6 |
0.51 |
NS |
50 |
II |
4-6 |
1179 |
41 |
3.48 |
<0.01 |
50 |
III |
7-9 |
156 |
2 |
1.28 |
NS |
50 |
I-III |
0-9 |
2520 |
49 |
1.94 |
<0.01 |
NS = not statistically significant.
Note 1: The relatively low mutation frequency in brood II at 0 mM (of 0.06%) was consistent with historical control data.
Note 2: No tabular data was presented for the test chemical at 10 mM but it was concluded by the authors to be non-mutagenic in the text.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Data available from various sources was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:
Ames assay:
Ames assay was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the given test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.
In vitro mammalian chromosome aberration study:
In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.25 mg/ml for 24 and 48 hr. DMSO was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%.The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).
The test chemical was tested for clastogenic effects in Chinese hamster ovary (CHO) cells at 0 (solvent control) and 25 mg/ml, with and without metabolic activation (S9). The top dose was half the dose that induced mitotic inhibition which was defined as one metaphase or less in 6000 CHO cells. 200 metaphases per sample were scored for chromosomal aberrations. The results were presented as the frequency of metaphases with chromosomal aberrations, the number of chromatid breaks per cell, and the number of chromatid exchanges per cell. No significant effect was observed following treatment with the test chemical compared to solvent control data. No positive controls were used, however, several other agents that were evaluated in the study tested clearly positive for clastogenic effects without and/or with metabolic activation. This is taken as an indication that the assay was valid for the detection of clastogenic effects in CHO cells.
In vitro gene mutation study in mammalian cells
An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, chemical was administered to CHO cells for 3 hrs at the dose levels of 0.0625, 0.125, 0.25 or 0.5 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. N-ethyl-N-nitrosourea (ENU)and7,12-dimethylbenz(a) anthraceneserved as positive.The positive control used in absence of metabolic activation (i.e.N-ethyl-N-nitrosourea) produced a significant increase in the number of revertant colonies whereas the positive control used in presence of metabolic activation (i.e. 7,12 -dimethylbenz(a)anthracene) did not. Without metabolic activation, the test chemical tested negative for mutagenicity in CHO cells. No conclusions could be reached regarding the mutagenicity of the test chemical in CHO cells in the presence of the metabolic activation system.
Justification for classification or non-classification
Sodium salicylate has tested negative for mutagenicity in bacteria in a study according to OECD Test Guideline 471. Read-across chemical No.: 1 has tested negative for clastogenic effects in CHO cells with and without metabolic activation. In mice, treatment with sodium salicylate produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to solvent control data. This weak response was attributed to the ability of the chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the same study. In an in vitro mammalian gene mutation assay, treatment with sodium salicylate tested negative for mutagenicity in CHO cells in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity of the chemical in the presence of metabolic activation due to invalid positive control data. However, in a sex-linked recessive lethal test in Drosophila melanogaster, three days of dosing with read-across chemical No.: 2 at LD50 failed to produce any significant increase in the frequency of sex-linked recessive lethal mutations. Therefore, by weight of evidence, sodium salicylate is regarded to be classified as Not Classified for Germ cell mutagenicity.
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