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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 471
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate
Test concentrations with justification for top dose:
50, 158, 500, 1580, & 5000.0 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 aminoanthramine for all the strains in the absence of S9, sodium azide-S9TA 100 & TA 1535, 2NF -S9 in TA 100, 2AA -S9 in TA 1537
Details on test system and experimental conditions:
Type: Ames test

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 min
- Incubation period: 48 hours

NUMBER OF REPLICATIONS:
3 replicates per dose

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn evaluation

Evaluation criteria:
Concentrations of the test chemical which are overtly toxic are easily visualized as showing no bacterial growth on the plate (i.e. absence of background lawn). Lower levels of toxicity may be seen as a thin or sparse bacterial lawn, a reduction in the number of revertants, or the appearance of microcolonies (overgrown background lawn). Positive and negative controls were run concurrently with the test chemical, and appropriate responses for these controls are prerequesites for evaluating the response of the bacteria to the test chemical.

A test chemical is considered a bacterial mutagen if both the mean number of revertant colonies observed is at least three times higher than the mean of the negative (solvent) control and at the same time it produces a dose response relationship over several concentrations. If a chemical produces reproducible reversion rates in excess of 3x over background, but no definitive dose response relationship, it is considered to be a presumptive bacterial mutagen. If a chemical produces reproducible reversion rates greater than 2x but less than 3x over the negative controls, the results are considered to be equivocal or inconclusive. Test substances failing to meet the above criteria are considered non-mutagenic in this system.

Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:


RANGE-FINDING/SCREENING STUDIES: was conducted
COMPARISON WITH HISTORICAL CONTROL DATA: was done
ADDITIONAL INFORMATION ON CYTOTOXICITY:no cytotoxicity was observed
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

see the attached word doc for tables.

Conclusions:
Interpretation of results
negative

DOWANOL TPnB did not induce a mutagenic response in any of the tester strains as judged by the frequency of histidine-independent (his+) revertants. Hence, DOWANOL TPnB was  classified as negative in the Ames test under the experimental conditions used.
Executive summary:

TriPropylene glycol- n-butyl ether was evaluated in the Salmonella/mammalian-microsome bacterial mutagenicity assay (Ames test) using a pre-incubation modification of the standard assay. The test was conducted both in the presence and in the absence of externally supplied metabolic activation system. The test was conducted using Salmonella typhimurium bacetrial tester strains TA98, TA100, TA1535 and TA1537. The test agent was initially assayed in TA100 at concentrations of 0 (solvent control), 5.0, 15.8, 50.0, 158.0, 500.0, 1580.0 and 5000.0 micrograms/plate. The test material was assayed at least two times in each tester strain up to a maximum concentration of 5000 ug/plate.

In addition to the mutation in the histidine operon, these tester strains contain other mutations that increase their ability to detect mutagens.

Based upon the results in TA100, the top five concentrations were repeated in TA98, TA100, TA1535 and TA1537. The assay was repeated independently at the same five concentrations using all four tester strains. The test material did not induce a mutagenic response in any of the tester strains in either of the assays as judged by the frequencies of histidine-independent (his+) revertants. Hence, the test material was classified as negative in the under the experimental conditions used.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
June 1994-March 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 476
Justification for type of information:
Please refer to category document.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 nutrient mix (GIBCO) supplemeted with 5% heat-inactivated dialyzed fetal bovine serum (GIBCO), 25 mM HEPES (GIBCO) and antibiotics-antimycotics (GIBCO).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9 activation system
Test concentrations with justification for top dose:
279 - 5000 microG/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
20-methylcolanthrene with S9 Migrated to IUCLID6: without S9
Details on test system and experimental conditions:
Stock cells were grown in Ham's serum containing F-12 nutrient mix, which also contained hypoxanthine required by the cell line.  Cells in logarithmic growth phase, grown to a density of 3 x 106 cells/T-25 flask (for gene mutation assay; 1 x 106 cells/T-25 flask for toxicity assay), were trypsinized and plated.  Approximately 24 hours after plating, medium was replaced with 1) fresh medium without serum, 2) the test material, negative control solvent (DMSO), or positive controls, 3) with or without S-9 supernatant.  Cells incubated with the test material at 37°C for approximately 4 hours, then cells were washed with phosphate-buffered saline to terminate treatment.  Subsequently, cultures were trypsinized and re-plated at a density of 1 x 106 cells per 100 mm dish (2 dishes per replicate) in medium still containing hypoxanthine for 6 to 8 days for phenotypic expression.  At the end of the 6-8 day expression period, cultures were trypsinized and plated at a density of 2 x 105 cells/100 mm dish (10 dishes/replicate) in the selection medium (Ham's 12 without hypoxanthine and with 6-thioguanine) for selection of HGPRT- mutants.  During this selection period, dishes were incubated at 37°C for 8-10 days to allow for colony formation.  At the end of this time, the cells were fixed with methanol and stained with crystal violet.  Mutant frequency was determined from the number of colonies formed in the dishes, taking into account cloning efficiency. 
Evaluation criteria:
20-Methylcholanthrene (4 ug/ml) was the positive control agent with S-9 and ethylmethanesulfonate (621 ug/ml) was the positive control agent without S-9.  DMSO at 1% was the negative control.
Statistics:
The frequency of mutants were evaluated using a weighted analysis of variance; weights are derived from the inverse of the mutation frequency variance. A linear trend test and lack of fit test is employed (alpha=0.05), as an omnibus test to compare treated groups to the negative control. Dunnett's t-test is conducted comparing each treated group and the positive control to the negative control, if there is a significant increasing trend or a significant lack of fit. An additional comparison of the positive control to the negative control is conducted using a linear contrast statement.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the toxicity assay, doses up to and including 1250 ug DPnB/ml culture medium were without effect.  Without S-9, doses of 2500 and 5000 ug/ml showed Relative Cell Survival (RCS) of 61% and 56%, respectively.  With S-9, toxicity occurred only at 5000 ug/ml (38%).  In the mutation assay itself, toxicity was less as evidenced by higher RCS at higher dose levels. In the first mutation assay, doses ranged slightly below target of 5000 ug/ml.  Specifically doses ranged from 279 to 4467 ug/ml.  No toxicity was seen without S-9 at the highest dose level.  With S-9, toxicity was seen at the highest dose only (4467 ug/ml) with 48% RCS in one replicate and 36% in another. In this first mutation assay, mutation frequencies were not different from controls either with or without S-9 metabolic activation.  Negative and positive controls fell within laboratory historical limits. In the second mutation assay, doses ranged from 312 up to 5000 ug/ml.  Without S-9, cytotoxicity occurred only at the highest dose tested, 84% in one duplicate and 66% in the second.  With S-9, toxicity was seen at 2500 ug/ml (75% & 90%) and 5000 ug/ml (62% & 80%).  In this second assay, mutation frequencies were not different from controls either with or without S-9 metabolic activation.  Negative and positive controls fell within laboratory historical limits.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative with and without metabolic activation system

DPnB is not mutagenic in the CHO/HGPRT forward mutation assay.
Executive summary:

Stock cells were grown in Ham's serum containing F-12 nutrient mix, which also contained hypoxanthine required by the cell line.  Cells in logarithmic growth phase, grown to a density of 3 x 106 cells/T-25 flask (for gene mutation assay; 1 x 106 cells/T-25 flask for toxicity assay), were trypsinized and plated.  Approximately 24 hours after plating, medium was replaced with 1) fresh medium without serum, 2) the test material, negative control solvent (DMSO), or positive controls, 3) with or without S-9 supernatant.  Cells incubated with the test material at 37°C for approximately 4 hours, then cells were washed with phosphate-buffered saline to terminate treatment. Subsequently, cultures were trypsinized and re-plated at a density of 1 x 106 cells per 100 mm dish (2 dishes per replicate) in medium still containing hypoxanthine for 6 to 8 days for phenotypic expression.  At the end of the 6-8 day expression period, cultures were trypsinized and plated at a density of 2 x 105 cells/100 mm dish (10 dishes/replicate) in the selection medium (Ham's 12 without hypoxanthine and with 6-thioguanine) for selection of HGPRT- mutants.  During this selection period, dishes were incubated at 37°C for 8-10 days to allow for colony formation.  At the end of this time, the cells were fixed with methanol and stained with crystal violet.  Mutant frequency was determined from the number of colonies formed in the dishes, taking into account cloning efficiency. 

20-Methylcholanthrene (4 ug/ml) was the positive control agent with S-9 and ethylmethanesulfonate (621 ug/ml) was the positive control agent without S-9.  DMSO at 1% was the negative control.

In the toxicity assay, doses up to and including 1250 ug DPnB/ml culture medium were without effect.  Without S-9, doses of 2500 and 5000 ug/ml showed Relative Cell Survival (RCS) of 61% and 56%, respectively.  With S-9, toxicity
occurred only at 5000 ug/ml (38%).  In the mutation assay itself, toxicity was less as evidenced by higher RCS at
higher dose levels.

In the first mutation assay, doses ranged slightly below target of 5000 ug/ml.  Specifically doses ranged from 279 to
4467 ug/ml.  No toxicity was seen without S-9 at the highest dose level.  With S-9, toxicity was seen at the highest dose
only (4467 ug/ml) with 48% RCS in one replicate and 36% in another. In this first mutation assay, mutation frequencies
were not different from controls either with or without S-9 metabolic activation.  Negative and positive controls fell
within laboratory historical limits.

In the second mutation assay, doses ranged from 312 up to 5000 ug/ml.  Without S-9, cytotoxicity occurred only at the
highest dose tested, 84% in one duplicate and 66% in the second.  With S-9, toxicity was seen at 2500 ug/ml (75% &
90%) and 5000 ug/ml (62% & 80%).  In this second assay, mutation frequencies were not different from controls either
with or without S-9 metabolic activation.  Negative and positive controls fell within laboratory historical limits.

In conclusion, DPnB is not mutagenic in the CHO/HGPRT forward mutation assay.

This study was identified as key for this toxicity endpoint because of the methods followed (which were well documented
in the report).  The report included GLP and Quality Assurance statements, signed by the Study Director and Head
of the QA Unit, respectively.  The cell line used, test substance concentrations and dose spacing (4 dose levels
including negative control, with highest being 5000 ug/ml), time exposed to the test and control agents, positive control agents used, metabolic activation system, number of replicates, the number of cells scored, and scoring criteria
all followed or exceeded guidance as specified in OECD Guideline 476 "In Vitro Mammalian Cell Gene Mutation Test." 
The positive control agents gave the expected results showing that the cell line was responsive to forward mutation insult.

Unlike the cytogenietics studies conducted by Dow that used no vehicle solvent, DMSO was used as a diluent for DPnB in
this assay.  DMSO served as the negative control at 1% concentration within the media.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
03/2009-09/2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 473.
Justification for type of information:
Please refer to category document.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Sprague-Dawley derived CD IGS (outbred Crl:CD(SD)) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier. Blood samples were collected by cardiac puncture, following euthanasia with carbon dioxide. In the assays, 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% 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), 25 mM HEPES (GIBCO), 30 μg/ml PHA-P (HA16, Murex Diagnostics Ltd., Dartford, England), and an additional 2 mM Lglutamine (GIBCO). Cultures were initiated by inoculating approximately 0.5 ml of whole blood/5 ml of culture medium. Cultures were set up in duplicate at each dose level in sterile disposable tissue culture flasks and incubated at 37°C.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate prepared from Aroclor 1254 treated (500 mg/kg BW) male Sprague- Dawley rats.
Test concentrations with justification for top dose:
initial assay (4h with S9): 78.1, 156.3, 312.5, 625, 1250, 2500, and 5000 μg/ml
initial assay (24h without S9): 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500, and 5000 μg/ml
repeat assay (4 h with and without S9): 200, 400, 600, 800, 1000, 1400, 1600, and 2000 μg/ml
Vehicle / solvent:
The test material was first dissolved in DMSO and further diluted (1:100) with the treatment medium to obtain the desired concentrations.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Mitomycin C (without S9); cyclophosphamide monohydrate (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 4 and 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: One hundred metaphases/replicate were examined from coded slides for structural abnormalities

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the solvent controls. The aberration frequency in the solvent control should be within reasonable limits of the laboratory historical values. A test chemical will be considered positive in this assay if it induces a significant, doserelated increase in the frequency of cells with aberrations and the incident of aberrant cells and cultures is outside the recent historical solvent control range.
Statistics:
The proportions of cells with aberrations (excluding gaps) were 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 global hypothesis of no difference in the average number of cells with aberrations among the dose groups. An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, versus a one-sided increasing alternative, pairwise tests (i.e. control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, again versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed. Polyploid cells were analyzed by the Fisher Exact probability test. The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha = 0.05. The data was analyzed separately based on the presence or absence of S9 and based on the exposure time.
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no appreciable change in either the pH or osmolality at this concentration as compared to the culture medium with solvent alone (culture medium with the test material, pH = 7.35, osmolality = 389 mOsm/kg H20; culture medium with 1% DMSO,
pH = 7.35, osmolality = 400 mOsm/kg H20).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

It was concluded that under the experimental conditions used, DOWANOL™ TPNB-H Glycol Ether was non-genotoxic in this in vitro chromosomal aberration test.
Executive summary:

DOWANOLTM TPNB-H Glycol Ether (reaction mass of α-butyl-ω-hydroxy-poly(oxy(methyl- 1,2-ethanediyl)) (polypropylene glycol monobutyl ether) and (2-(2- butoxymethylethoxy)methylethoxy)-propanol (tripropylene glycol monobutyl ether) was evaluated in an in vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (solvent control) to 3250 μg per ml of culture medium. The duration of treatment was 4 or 24 hours without S9 and 4 hours with S9. The highest concentration was based on toxicity of the test material to the lymphocyte cultures. Based upon the mitotic indices, cultures treated for 4 hours with targeted concentrations of 0 (solvent control), 600, 800, and 1400 μg/ml in the absence of S9 activation and 0 (solvent control), 207, 807, and 1620 μg/ml in the presence of S9 activation were selected for determining the incidence of chromosomal aberrations and incidence of polyploidy. Cultures treated for 24 hours in the absence of S9 with 0 (solvent control), 99, 207, and 419 μg/ml were selected for determining the incidence of chromosomal aberrations. In a confirmatory assay performed with S9, targeted concentrations of 0 (solvent control), 400, 800, and 1000 μg/ml were selected for determining the incidence of chromosomal aberrations and incidence of polyploidy. There were no statistically significant increases in the frequencies of cells with aberrations in any of the treatments in either the absence or presence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of abnormal cells in all assays. Based upon these results, DOWANOL™ TPNB-H Glycol Ether was considered to be non-genotoxic in this in vitro chromosomal aberration assay utilizing rat lymphocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
05/2009-11/2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 476.
Justification for type of information:
Please refer to category document.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT locus of Chinese hamster ovary (CHO) cells in culture
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. Stock cultures were stored at about -80°C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture labware under standard conditions of approximately 5% CO2 in air at 37C in a humidified incubator. The cells were routinely maintained in Ham's F-12 nutrient mix (GIBCO, Grand Island, New York) supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 25 µg/ml; GIBCO), and an additional 2 mM L-glutamine (GIBCO). The selection medium used for the detection of HGPRT- mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6 thioguanine (GIBCO), and 5% serum and the above-mentioned antibiotics.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
preliminary toxicity assay (with and without S9): 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500, and 5000 µg/ml
initial mutagenicity assay (with and without S9): 500, 1000, 1250, 1500, 1750, 2000, 2250, and 2500 µg/ml
confirmatory mutagenicity assay (with S9): 1400, 1600, 1800, 1900, 2000, 2100, 2200, 2300 and 2400 µg/ml
confirmatory mutagenicity assay (without S9): 1000, 1200, 1400, 1500, 1600, 1700, 1800, 1900 and 2000 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate (without S9); 20-methylcholanthrene (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7-9 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 cells/dish

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the solvent controls. An additional criteria, was that the mutant frequency in the solvent controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the solvent controls.
Statistics:
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutation frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was used as an estimate of variance. If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted, comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The pH and osmolality of treatment medium containing approximately 5036 µg/ml (approximately the 5000 µg/ml limit concentration) 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). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in In Vitro genotoxicity assays. There was slight change in the osmolality at this concentration as compared to the culture medium with solvent alone which was deemed inconsequential to the conduct of this study and there was no appreciable change in the pH as compared to the culture medium with solvent alone (culture medium with the test material, pH = 7.30, osmolality = 336 mOsm/kgH2O; culture medium with 1% DMSO, pH = 7.30, osmolality = 397 mOsm/kgH2O).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

The results of the In Vitro Chinese hamster ovary cell/hypoxanthine-quanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with DOWANOL TPNB-H glycol ether indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

DOWANOL TPNB-H Glycol Ether (reaction mass of α-butyl-ω-hydroxy-poly(oxy(methyl-1,2-ethanediyl)) (polypropylene glycol monobutyl ether) and (2-(2-butoxymethylethoxy)methylethoxy)-propanol (tripropylene glycol monobutyl ether)) was evaluated in the In Vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation (S9) system. The concentrations ranged from 500 to 2500 µg/ml in the absence and presence of S9. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e. dimethyl sulfoxide). The results of the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with DOWANOLTPNB-H glycol etherindicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 474
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: charles River, Kingston,NY
- Age at study initiation: 8 week old
- Assigned to test groups randomly: yes
- Housing:singly
- Diet - ad libitum)
- Water - ad libitum)
- Acclimation period:one week


ENVIRONMENTAL CONDITIONS
adequate environmental condition


Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: single oral dose


DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:
Frequency of treatment:
single
Post exposure period:
groups of animals were sacrificed at 3 intervals 24, 48, and 72h after treatment.
Remarks:
Doses / Concentrations:
0, 187.5, 625.0, and 1875.0 mg/kg bw
Basis:

No. of animals per sex per dose:
5 animals per sex per dose per treatment time
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): historical data
- Route of administration: oral
- Doses / concentrations:120mg/kg body weight
Tissues and cell types examined:
bone marrow cells for PCE
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on the dose range finding test
TREATMENT AND SAMPLING TIMES: at 24, 48, 72h

DETAILS OF SLIDE PREPARATION: At the end of the sacrificed intervals following dosing, the animals were sacrificed by cervical dislocation. Bone morrow samples were obtained from the femurs. Bone marrow was aspirated into fetal bovine serum. After aspiration, the contents of the syringe were transferred into a 1.5 ml centrifuge tube containing 0.5 ml of serum. The cells were resuspended in serum by gentle aspiration using the syringe and needle to centrifuge. After centrifugation supernatant was discarded leaving a small amount of serum covering the pellet. The cell pellet was resuspended using a disposable transfer pipet. Cell smear were prepared on microscope slides using small portions of the cell suspension. The cells were allowed to air dry, fixed in methanol and stained in 5% Giemsa.

METHOD OF ANALYSIS: The slides were coded and scored blindly. One thousand PCE were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were identified as darkly stained bodies with sharp contours and varying shapes such as round, almond or ring (Schmid, 1976). The ratio of PCE-NCE in the bone marrow was determined by examining 1000 erythrocytes. The ratio was expressed as PCEx100/PCE+NCE.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in the number of micronucleated cells or a clear increase in the number of micronucleated cells in a single dose group at a single sampling time. Biological relevance of the results should be considered first. Statistical methods may be used as an aid in evaluating the test results.
Statistical significance should not be the only determining factor for a positive response. Equivocal results should be clarified by further testing preferably using a modification of experimental conditions.
A test substance for which the results do not meet the above criteria is considered nonmutagenic
in this test.
Although most experiments will give clearly positive or negative results, in rare cases the data set will preclude making a definite judgement about the activity of the test substance. Results, may remain equivocal or questionable regardless of the number of times the experiment is repeated.
Positive results in the micronucleus test indicate that a substance induces micronuclei which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. Negative results indicate that, under the test conditions, the test substance does not produce micronuclei in the immature erythrocytes of the test species.
The likelihood that the test substance or its metabolites reach the general circulation or specifically the target tissue (e.g. systemic toxicity) should be discussed.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
There were no significant increases in the frequencies of MN-PCE in groups treated with DOWANOL TPnB compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under the experimental conditions used, DOWANOL TPnB was considered negative in the mouse bone marrow micronucleus test.
Executive summary:

DOWANOL TPnB was evaluated in the mouse bone marrow micronucleus test. The micronucleus test is capable of
detecting agents causing chromosomal aberrations and spindle malfunction.  DOWANOL TPnB was mixed with corn oil and administered to CD-1 

(ICR) BR  mice by single oral gavage at dose levels of 0 (negative control), 187.5,  625.0, and 1875.0 mg/kg body weight. 

Groups of animals were sacrificed at three intervals, viz., 24, 48, and 72 hours after treatment. Mice treated with 120mg/kg body weight cyclophosphamide and sacrificed at 24 hour served as positive controls. There were five animals
per sex per dose level per sacrifice time. At the end of the sacrificed intervals following dosing, the animals were sacrificed by cervical dislocation. Bone morrow samples were obtained from the femurs. Bone marrow was aspirated into fetal bovine serum. After aspiration, the contents of the syringe were transferred into a 1.5 ml centrifuge tube containing 0.5 ml of serum. The cells were resuspended in serum by gentle aspiration using the syringe and needle to centrifuge. After centrifugation supernatant was discarded leaving a small amount of serum covering the pellet. The cell pellet was resuspended using a disposable transfer pipet. Cell smear were prepared on microscope slides using small portions of the cell suspension. The cells were allowed to air dry, fixed in methanol and stained in 5% Giemsa.

The slides were coded and scored blindly. One thousand PCE were examined from each animal and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. Micronuclei were identified as darkly stained bodies with sharp contours and varying shapes such as round, almond or ring (Schmid, 1976). The ratio of PCE-NCE in the bone marrow was determined by examining 1000 erythrocytes. The ratio was expressed as PCEx100/PCE+NCE.

Based on study results there were no significant increases in the frequencies of MN-PCE in groups treated with DOWANOL TPnB compared to negative controls. The positive control mice showed significant increases in MN-PCE. Hence, under experimental conditions used, DOWANOL TPnB was considered negative in the mouse bone marrow micronucleus test.

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

Additional information

All genotoxicity studies are reliable without restrictions as they were conducted under GLP and according to OECD guidelines. The in vitro gene mutation study in bacteria as well as the in vivo micronucleus test, both conducted with tripropylene glycol butyl ether, were clearly negative. In addition, read-across data for mutagenicity in mammalian cells is available from the structural analogues dipropylene glycol butyl ether and tripropylene glycol butyl ether highers. Both analogues showed clearly negative results in the mutagenicity assays.

The justification for the use of read across is found int he category document.


Short description of key information:
GLP-studies according to OECD guidelines 471 and 474 are available for tripropylene glycol butyl ether. In addition GLP-studies according to OECD guideline 476 are available for the structural analogues dipropylene glycol butyl ether and tripropylene glycol butyl ether highers.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

All in vitro and in vivo genotoxicity studies for mutagenicity and cytogenicity endpoints were clearly negative. Hence, no classification for genotoxicity applies to tripropylene glycol butyl ether.