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Genetic toxicity: in vitro

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to test guidlines and in accordance with GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Remarks:
Not specified in report
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
Remarks:
Not specified in report
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2000
Deviations:
no
Remarks:
Not specified in report
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Source: Union Carbide Corporation, a Subsidiary of The Dow Chemical Company
Lot Number: 7897
Purity/Characterizarion: 98.3% average area percent (the sum of two isomers) as determined by gas
chromatography with thermal conductivity detection (GC/TCD. The water content
was determined to be 0.09% using a Karl Fischer coulometric titration
(VanderKamp, et al, 2002).
Molecular formula: C9H20O
Molecular Weight: 144

Method

Target gene:
cultured rat lymphocytes
Species / strain
Species / strain / cell type:
lymphocytes: Rat
Details on mammalian cell type (if applicable):
Blood samples were collected from male CD rats (outbred Crl:CDÒ(SD) IGS BR strain purchased from Charles River, Portage, MI), aged approximately 13 weeks in Assay A1 and B1 and 14 weeks in Assay B2. Blood samples were collected by cardiac puncture, following euthanasia with carbon
dioxide, from 3 rats in Assay A1 and 2 rats in Assays B1 and B2. In each assay, blood samples from individual rats were pooled and whole blood
cultures were set up in RPMI 1640 medium (with 25 mM HEPES, GIBCO, Grand Island, New York) supplemented with 10% dialyzed heat-inactivated
fetal bovine serum (GIBCO), antibiotics and antimycotics (Fungizone 0.25 μg/ml; penicillin G, 100 U/ml; and streptomycin sulfate, 0.1 mg/ml; GIBCO), 30 μg/ml PHA (HA16, Murex Diagnostics Ltd., Dartford, England), and an additional 2 mM L-glutamine (GIBCO). Cultures were initiated by inoculatingapproximately 0.5 ml of whole blood/5 ml of culture medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 Liver Cells
Test concentrations with justification for top dose:
0, 100, 200, 300, 400, 600, 800, 1000, 1200, and 1440 ug/ml
Vehicle / solvent:
Diisobutyl carbinol has a low water solubility of approximately 445 μg/ml at 25°C. Therefore, the test material was first dissolved in dimethyl
sulfoxide (DMSO, Sigma) and further diluted (1:100) with the treatment medium to obtain the desired concentrations. All prepared stock solutions
were submitted to the Analytical Chemistry Laboratory of the testing facility for the verification of test material concentrations. MMC and CP were
dissolved directly in treatment medium. The treatment medium was RPMI 1640 with HEPES and antibiotics, without the serum and the PHA.
Controls
Untreated negative controls:
yes
Remarks:
solvent (DMSO)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Remarks:
Mitomycin C- non-activation assays
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: activation assays
Details on test system and experimental conditions:
Treatment Procedure without Metabolic Activation:
Approximately fo rty-eight hours after initiation of the cultures, the cell suspension was dispensed into 15 ml sterile centrifuge tubes (approximately 5.5 ml/tube, two cultures per dose level). The cells were sedimented by centrifugation and the culture medium removed and saved. The cells were
exposed to medium (RPMI 1640, HEPES, and antibiotics) containing the test or positive or negative control treatments for approximately 4 hours at
37°C and the exposure was terminated by washing the cells with culture medium. The cells were then placed in individual sterile disposable tissue
culture flasks (T-25) along with approximately 4.5 ml of the original culture medium until the time of harvest. The cultures were harvested at
approximately 24 hours after treatment initiation (i.e., approximately 20 hours after treatment termination).

A second set of cultures was treated with the test material continuously for 24 hours (approximately 1.5 normal cell cycle length). Stock solutions of the treatments were added directly to the culture flasks at 48 hours after initiation of the cultures and thesecultures were harvested 24 hours later.

Treatment Procedure using Metabolic Activation:
Approximately 48 hours after initiation of the cultures, the cell suspension was dispensed into sterile disposable centrifuge tubes. The cells were sedimented by centrifugation and theculture medium removed and saved. The cells were exposed to medium (RPMI 1640, HEPES, antibiotics, and S-9)
containing the test and positive and negative control treatments for approximately 4 hours at 37°C and the exposure was terminated by washing
the cells with culture medium (without serum and PHA). The cells were then placed in individual sterile disposable tissue culture flasks (T-25) along
with approximately 4.5 ml of the original culture media until the time of harvest (i.e., approximately 20 hours after treatment termination).
Evaluation criteria:
Colcemid was added approximately 3 hours prior to harvest at a final concentration of 0.2 μg/ml. The cells were swollen by hypotonic treatment
(0.075 M KCl), fixed with methanol:acetic acid (3:1), dropped on microscope slides, and stained in Giemsa. All slides were coded prior to evaluation.
Mitotic indices were determined as the number of cells in metaphase among 1000 cells/replicate and expressed as percentages. One hundred
metaphases/replicate were examined, where possible, from coded slides at each selected concentration of the test chemical and the negative controls (a total of 200 cells/treatment) for structural abnormalities (Buckton and Evans, 1973; Sinha et al., 1984; Gollapudi et al.,1986). In the positive
control cultures, 50 metaphases/replicate (a total of 100 cells/treatment) were examined for abnormalities unless otherwise indicated in the tables.
The microscopic coordinates of those metaphases containing aberrations were recorded. Only those metaphases that contained 42 + 2 centromeres were scored with the exception of cells with multiple aberrations, in which case accurate counts of the chromosomes were not always possible.
Structural chromosomal abnormalities that were counted included chromatid and chromosome gaps, chromatid breaks and exchanges, chromosoe breaks and exchanges, and miscellaneous (chromosomal disintegration, chromosomal pulverization, etc.). Those cells having five or more aberrations/cell were classified as cells with multiple aberrations. Gaps were not included in calculations of total cytogenetic aberrations. In addition, one
hundred metaphases/replicate were examined forthe incidence of polyploidy. The data were used to calculate the following parameters:
Statistics:
The proportion of cells with aberrations (excluding gaps) was compared by the following statistical methods. At each dose level, data from the
replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of aberrant cells. An overall Chi-square statistic,
based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the two global hypotheses of (1) no
differences in average number of cells with aberrations among the dose groups, and (2) no linear trend of increasing number of cells with aberrations with increasing dose (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If either statistic was
found to be significant at a = 0.05 versus a one-sided increasing alternative, pairwise tests (i.e., control vs. treatment) were performed at each
dose level and evaluated at a = 0.05 again versus a one-sided alternative. Polyploid cells were analyzed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells was pooled across replicates for the analysis and evaluated at a = 0.05. The data were analyzed separately based on the presence or absence of S-9 and based on the exposure time. For a test to be acceptable, the chromosomal aberratio n frequency in the
positive control cultures should be significantly higher than the negative controls. The aberration frequency in the negative control should be within reasonable limits of the laboratory historical values. A test chemical is considered positive in this assay if it induces a significant dose-related
and reproducible increase in the frequency of cells with aberrations.

Results and discussion

Test results
Species / strain:
lymphocytes: rat peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

pH and Osmolality

The pH and osmolality of treatment medium containing approximately 1500 (limit dose of approximately 10 mM) and 750 μg/ml of the test material and medium containing 1%DMSO were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada,Colorado) and an Osmette A freezing point osmometer (Precision Systems, Inc., Natick,

Massachusetts), respectively. There was no appreciable change in either the pH orosmolality at these concentrations as compared to the culture medium with solvent alone (culture medium with 1500 μg/ml of the test material, pH = 7.41, osmolality = 403 mOsm/kg H20; culture medium with 750 μg/ml of the test material, pH = 7.34, osmolality= 418 mOsm/kg H2O; culture medium with 1% DMSO, pH = 7.31, osmolality = 432 mOsm/kg H20).

Assay A1

In the initial assay, cultures were treated with the test material in the absence and presence of S-9 activation for 4 hr at concentrations of 22.5, 45, 90, 180, 360, 720, and 1440 μg/ml (Tables 4A and 4B). Cultures were also treated continuously for 24 hr in the absence of S-9 with the above concentrations plus an additional lower concentration of 11.25 μg/ml (Table 4C). The highest concentration evaluated was based upon the limit dose of approximately 10 mM. The analytically detected concentrations of the test material in the stock solutions varied from 92 to 112% of the target (Table 1). Without metabolic activation (4 hr treatment), moderate toxicity was observed (30 and 39% reduction in mitotic index, MI) in cultures treated with 720 and 1440 μg/ml, respectively (Table 4A). The remaining cultures treated at the lower concentrations (22.5, 45, 90, 180, and 360 μg/ml) had reductions in mitotic indices ranging from 0 to 19%. In the presence of S-9 activation, cultures treated with the two higher concentrations (720 and 1440 mg/ml) had no mitoses and cultures treated with 360 μg/ml showed a 60% reduction in MI (Table

4B). The remaining cultures (22.5, 45, 90, and 180 mg/ml) had reductions in mitotic indices ranging from 0 to 15%. Cultures treated for 24 hr without S-9 activation had no mitosis at concentrations of 360, 720, and 1440 μg/ml (Table 4C). Cultures treated with

180 μg/ml had a 95% reduction in MI. The remaining cultures treated with concentrations of 11.25, 22.5, 45, and 90 μg/ml had reductions in mitotic indices of 13, 25, 18, and 52%, respectively. Based upon these results, the following cultures were selected for the determination of chromosomal aberrations and incidence of polyploidy: 360, 720, and 1440 mg/ml without S-9, 4 hr treatment; 90, 180, and 360 mg/ml with S-9, 4 hr treatment; 22.5, 45 and 90 mg/ml without S-9, 24 hr treatment. Among the cultures treated with the positive control chemicals for 4 hr, 0.5 mg/ml of MMC and 6 mg/ml of CP were selected for the evaluation of aberrations in the absence and presence of S-9, respectively. Cultures treated with 0.05 μg/ml MMC were selected for evaluation to serve as the positive control for the 24 hr in the absence of S-9. There were no significant increases in the incidence of polyploid cells in the test material treated cultures as compared to the negative control values (Table 5). In the 4 hr non-activation assay (Table 6A), the frequency of cells with aberrations in the negative control was 0.5% and the corresponding values at treatment levels 360, 720 and

1440 μg/ml were 0.5, 1.5, and 3.5%, respectively. Statistical analyses of these data identified a significant trend test and a significant difference in the frequency of aberrant cells between the negative control (0.5%) and 1440 μg/ml (3.5%) but not the 360 and 720 μg/ml treatments (0.5 and 1.5%, respectively). However, the aberrant cell frequencies observed in the treated cultures were within the laboratory historical negative control values (Tables 11 and 12). Hence, it was provisionally concluded, pendingconfirmation in a repeat assay, that the statistically identified difference was of little biological significance. In the activation assay (Table 6B), cultures treated with the test material at concentrations of 90, 180 and 360 μg/ml had aberrant cell frequencies of 0.5, 2.5, and 3.0%, respectively, as compared to the negative control value of 2.5%. There were no statistically significant differences between cultures treated for 4 hr in the presence of S-9 activation and the corresponding negative control value and were within the laboratory historical background range (Tables 11 and 12). A second assay with treatment of cultures in the presence of S-9 was not considered necessary in this assay since the results of the initial test yielded clearly negative results. In the non-activation assay with 24 hr treatment with the test material, the frequencies of aberrant cells at concentrations of 22.5, 45 and 90 μg/ml, were 0, 1.5 and 1.5%, respectively, as compared to the negative control value of 0.5% (Table 6C). There were no statistically significant differences between test material treated and negative control values and all values were within the laboratory historical background range (Tables 11 and 12). Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemicals. Aberrant cell frequencies in MMC (without S-9, 4 hr treatment) CP (with S-9, 4 hr treatment) and MMC (without S-9, 24 hr treatment) cultures were 43% (Table 6A), 54% (Table 6B), and 27% (Table 6C), respectively.

Assay B1

In a repeat assay, to verify the statistical finding in Assay A1 (without S-9, 4 hr), rat lymphocyte cultures were treated for 4 hr in the absence of S-9 activation with 0, 100, 200, 300, 400, 600, 800, 1000, 1200, and 1440 μg/ml of the test material and cells were

harvested 20 hr after treatment termination. The analytically detected concentrations of the test material in the stock solutions varied from 106 to 120% of the target (Table 2). The mitotic indices are shown in Table 7. The highest concentration tested (1440 μg/ml)

showed excessive toxicity as evidenced by mitotic index reduction of 78%, and concentrations of 400, 600, 800 1000, and 1200 μg/ml had only a rare or no mitosis. The remaining cultures (100, 200, and 300 μg/ml) had reductions in mitotic indices of 0, 30

and 85%, respectively (Table 7). Due to the unusual dose response (which was attributed to solubility issues in subsequent experiments, see below) as well as, to excessive toxicity in this assay, the experiment was repeated (Assay B2).

Assay B2

In this repeat assay (4 hr treatment without S-9), cultures were treated for 4 hr in the absence of S-9 activation with 0, 100, 200, 300, 400, 600, 800, 1000, 1200, and 1440 μg/ml of the test material and cells were harvested 20 hr after treatment termination. The

analytically detected concentrations of the test material in the stock solutions varied from 101 to 126% of the target (Table 3). Again in this assay, there was an unusual dose response for mitotic inhibition. Cultures treated with 100, 200, and 1200 μg/ml had little to

no toxicity as evidenced by reductions in mitotic indices (Table 8). The remaining cultures treated with 300, 400, 600, 800, 1000, and 1440 μg/ml had reductions in mitotic indices of 61, 98, 100, 81, 45 and 37%, respectively. An oily layer of test material was observed in the treatment solutions at 800, 1000, 1200, and 1400 μg/ml, indicating that these concentrations exceeded the limit of solubility. The rise in the mitotic indices at the higher concentrations was attributed to the test material not being available to the cells because of the solubility problems. Based upon these results, cultures treated with 200, 300 and 1440 μg/ml were selected for the determination of chromosomal aberration frequencies and incidence of polyploidy. There were no significant increases in the incidence of polyploid cells in the test material treated cultures as compared to the negative control value (Table 9). The frequency of cells with aberrations in the negative control was 1.5% and the corresponding values at treatment levels 200, 300, and 1440 μg/ml were 2.5, 1.0, and 1.0%, respectively (Table 10). Statistical analyses of these data did not identify significant

differences between the negative control and any of the treated cultures in this assay. The frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range (Tables 11 and 12). Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. The aberrant cell frequency in MMC (0.5 μg/ml) treated cultures was 46.0% (Table 10).

Table C. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of Diisobutyl Carbinol in the

Absence of S-9 – Assay A1


            Treatment Duration: 4 Hours
            % MI
 Concentration Replicates    
 (ug/mL)  A  B  Average  RMI a
 Negative Control b 11.9   13.9  12.9  100.0
         
 22.5  13.4  13.8  13.6  105.4
 45  13.4  12.1  12.8  99.2
 90  16.5  12.8  14.7  114.0
 180  13.9  16.3  15.1  117.1
 360  9.8  10.9  10.4  80.6
 720  9.4  8.5  9.0  69.8
1140  9.1  6.5  7.8 60.5 
Positive Control c           
 0.5  10.5  10.2  10.4  80.6

a Relative Mitotic Index:   Average of Treated * 100   

Average of Negative Control
     

b 1% DMSO

c MMC

 

Table B. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of Diisobutyl Carbinol in the

Presence of S-9 – Assay A1


            Treatment Duration: 4 Hours
            % MI
 Concentration Replicates    
 (ug/mL)  A  B  Average  RMIa
 Negative Controlb  14.9 15.4 15.2   100.0
 22.5  13.9  14.1  14.0  92.1
 45  15.3  14.3  14.8  97.4
 90  13.1  12.7  12.9  84.9
 180  14.4  14.1  14.3  94.1
 360 5.1  7.0  6.1 40.1
 720  0.0  0.0  0.0  0.0
 1140  0.0  0.0  0.0  0.0
 Positive Controlc        
4.0  8.9   8.9  8.9  58.6
 6.0  3.9  8.0  6.0  39.5

aRelative Mitotic Index:   Average of Treated * 100  

Average of Negative Control
     

b1% DMSO

cMMC

Table C. Mitotic Indices (M.I.) of Rat Lymphocyte Cultures Treated with Various Concentrations of Diisobutyl Carbinol in the

Absence of S-9 – Assay A1


            Treatment Duration: 4 Hours
            % MI
 Concentration Replicates    
 (ug/mL)  A  B  Average  RMIa
 Negative Controlb  14.0 16.2  15.1  100.0
 11.25  13.3  12.8  13.1  86.8
 22.5  10.9  11.6  11.3  74.8
 45  12.0  12.7  12.4  82.1
90 8.6 5.7 7.2 47.7
180   1.2  0.4  0.8  5.3
 360  0.0  0.0  0.0  0.0
 720  0.0  0.0  0.0  0.0
 1140  0.0  0.0  0.0  0.0
 Positive Control c        

 4.0

 9.6  8.6  9.1  60.3
6.0  6.2  6.2  6.2  41.1
         
 a Relative Mitotic Index        

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Cultures treated with the positive control chemicals (i.e., mitomycin C without S-9 and cyclophosphamide with S-9) had significantly higher
incidences of abnormal cells in all assays. Hence, diisobutyl carbinol was considered to be non- genotoxic in this in vitro chromosomal
aberration assay utilizing rat lymphocytes.