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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:
May-November 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 471
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
none
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine mutation
Species / strain / cell type:
other: Salmonella/microsome test. Strains TA98, TA100, TA1535, TA1537, TA1538.
Details on mammalian cell type (if applicable):
n/a
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 
Test concentrations with justification for top dose:
100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
Dimethylsulphoxide (DMSO)
Negative solvent / vehicle controls:
yes
Remarks:
0.1 ml DMSO
Positive controls:
yes
Remarks:
see table 1 below for details
Details on test system and experimental conditions:
Frozen stock cultures of Salmonella typhimurium (from Bruce Ames, U California, Berkeley) were transferred to nutrient rich broth (Oxoid No. 3) and incubated at 37°C until reaching an optical density of 0.4 at 700 nm (or approximately 109 cells/ml).  This was done for each of the five tester strains (TA 98, 100, 1535, 1537, & 1538).  To 3 ml of liquefied top agar (45°C) was added: 1) 0.1 ml of fresh bacterial culture and 2) either 0.1 ml of a dilution of the test material in DMSO or 0.5 ml of a dilution of the test material in S-9 supernatant. The vortexed liquefied agar containing the test material was then poured into selective agar plates.  The plates were incubated in the dark at 37°C during which time histidine independent revertant colonies developed.  Colonies were counted with an Arteck Model 880 colony counter (or manually).  Results were considered positive if the number of colonies exceeded twice background for any of the strains at any dose and if a dose-response relationship was observed in any strain, with or without S-9 activation.  In addition the positive response had to be reproducible in a second experiment.  Results were considered negative if the revertant counts did not exceed background for any tester strain and the negative response is reproducible in a second experiment.
Evaluation criteria:
The validity of the assay was assessed by determining that 1) negative and positive control revertant counts fell within historical control counts and 2) toxicity did not interfere with interpretation of results.
Statistics:
Mean and standard deviation of the revertants were calculated.
Species / strain:
other: Strains TA98, TA100, TA1535, TA1537, TA1538.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DPnB was not toxic to the test organisms at concentrations up to and including 5000 µg/plate.  DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.  TA98 did show revertant counts that just met or barely exceeded twice background at two non-consecutive dose levels.  Because there was no dose-response and because this result was not repeated in a second assay, the results with TA98 were considered negative.  No other strain, with or without S-9 activation showed an increase in revertant counts.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

none

Conclusions:
Interpretation of results: negative

DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.
Executive summary:

Frozen stock cultures of Salmonella typhimurium (from Bruce Ames, U California, Berkeley) were transferred to nutrient
rich broth (Oxoid No. 3) and incubated at 37°C until reaching an optical density of 0.4 at 700 nm (or
approximately 109 cells/ml).  This was done for each of the five tester strains (TA 98, 100, 1535, 1537, & 1538).  To 3
ml of liquefied top agar (45°C) was added: 1) 0.1 ml of fresh bacterial culture and 2) either 0.1 ml of a dilution
of the test material in DMSO or 0.5 ml of a dilution of the test material in S-9 supernatant. The vortexed liquefied
agar containing the test material was then poured into selective agar plates.  The plates were incubated in the
dark at 37°C during which time histidine independent revertant colonies developed.  Colonies were counted with an
Arteck Model 880 colony counter (or manually).  Results were considered positive if the number of colonies exceeded twice
background for any of the strains at any dose and if a dose-response relationship was observed in any strain, with
or without S-9 activation.  In addition the positive response had to be reproducible in a second experiment. 
Results were considered negative if the revertant counts did not exceed background for any tester strain and the negative
response is reproducible in a second experiment.

The validity of the assay was assessed by determining that 1) negative and positive control revertant counts fell within historical control counts and 2) toxicity did not interfere with interpretation of results.

100, 333, 1000, 3330, 5000 µg/plate of DPnB was tested. DPnB was not toxic to the test organisms at concentrations up to and including 5000 µg/plate.  DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.  TA98 did show revertant counts that just met or barely exceeded twice background at two non-consecutive dose levels.  Because there was no dose-response and because this result was not repeated in a second assay, the results with TA98 were considered negative.  No other strain, with or without S-9 activation showed an increase in revertant counts.

The report did not specify which positive control agents were tested with each tester strain.  0.1 ml DMSO was used
as a vehicle to solubilize DPnB for the non-activation portion of the study.

In conclusion, DPnB did not cause mutations in the Ames plate assay with or without S-9 metabolic activation.

This study was identified as key for this toxicity endpoint because of the methods followed (which were 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 lines used, test substance concentrations and dose spacing (several dose levels
including negative control and a high dose of 5,000 µg/plate), time exposed to the test and control agents,
metabolic activation system, number of replicates, the number of plates scored, and scoring criteria all followed
or exceeded guidance as specified in OECD Guideline 471 "Bacterial Reverse Mutation Test".  The positive control
agents gave the expected results showing that the cell line was responsive to reverse mutation.

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:
April 1990-February 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study equivalent to OECD guideline 473
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Specific protocol guideline not specified (e.g., OECD Guideline No. 473: Genetic Toxicology, In Vitro Mammalian Cytogenetic Test). But the study was conducted in a method eqivalent or similar to OECD guideline473.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosome abberation
Species / strain / cell type:
other: CHO-K1, S1B cell line
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 nutrient mix medium containing 10% serum (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 factor
Test concentrations with justification for top dose:
0, 500, 1667, 5000 ug/ml of culture medium
Vehicle / solvent:
No vehicle used. Undiluted.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Cyclophosphomide (with S-9) Migrated to IUCLID6: without S-9
Details on test system and experimental conditions:
Chinese Hamster Ovary (CHO-K1, S1B) cells in logarithmic growth phase were tyrpsinized and plated in Ham's F-12 nutrient mix medium containing 10% serum at a density 2 x 105 cells/60 mm petri dish.  After approximately 24 hours, the medium was changed to new medium (2.5% serum) containing the test or control agents, with or without the S-9 supernatant metabolic activation system (from Aroclor 1254-induced rats).  Cells were exposed to test material (4 concentrations; 0, 500, 1667, or 5000 µDPnB/ml culture medium) and control agents for 4 hours at 37°C. 

At the end of 4 hours, cells were removed from the test and control agents by washing with phosphate-buffered saline and then maintained in culture medium (10% serum) until harvest. Duplicate cultures of each of the four dose levels of the test material-exposed cells and of the positive control agent-exposed cells were harvested 18 hours after exposure.  Two hours prior to harvest, cells were arrested in metaphase by addition of Colcemid.  At harvest, cells were trypsinized, swollen by hypotonic treatment, fixed on slides and stained with Giemsa.  Mitotic indices were computed by dividing the number of cells in metaphase by 500 cells examined (per replicate) and expressing this number as a percentage.  50 cells in metaphase per duplicate (total of 100) at each dose level (including positive controls) were examined for chromosomal aberrations.  Structural chromosomal abnormalities that were scored included chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and chromosomal disintegration.  Chromatid and chromosome gaps were not included in the number of total aberrations.
Evaluation criteria:
Positive control agents were: ethylmethanesulfonate (EMS) without the activation system and cyclophosphamide (CP) with the activation system. 
Statistics:
Descriptive and comparative statistics were performed for the abberation results.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Some toxicity was seen at highest dose tested (5000 ug/ml) with (53% survival compared to controls) and without (32% survival) S-9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
none
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.

Results are shown in the table below:

Dose Level     With/without    Cytotoxicity*    Aberrations
(ug/ml)         S-9
===========================================================
0 DPnB          +/-            Negative         Negative
500 DPnB        +/-            Negative         Negative
1667 DPnB       +/-            Negative         Negative
5000 DPnB       +/-            21% RCS          Negative
                               (both +/-)
1242 EMS         -             N/A              Positive
14 CP            +             N/A              Positive

* Cytotoxicity was greater at 5000 DPnB in this main assay than in the preliminary cytotoxicity assay.  No cytotoxicity
occurred at doses lower than 5,000 µg/ml in this main assay.

Conclusions:
Interpretation of results:
negative with and without metabolic activation

Dipropylene glycol n-butyl ether did not cause cytotoxicity or chromosomal aberrations under the conditions of this test.
Executive summary:

Chinese Hamster Ovary (CHO-K1, S1B) cells in logarithmic growth phase were tyrpsinized and plated in medium
containing 10% serum at a density 2 x 105 cells/60 mm petri dish.  After approximately 24 hours, the medium was changed
to new medium (2.5% serum) containing the test or control agents, with or without the S-9 supernatant metabolic
activation system (from Aroclor 1254-induced rats).  Cells were exposed to test material (4 concentrations; 0, 500,
1667, or 5000 µDPnB/ml culture medium) and control agents for 4 hours at 37°C.  Positive control agents were:
ethylmethanesulfonate (EMS) without the activation system and cyclophosphamide (CP) with the activation system.  At
the end of 4 hours, cells were removed from the test and control agents by washing with phosphate-buffered saline and
then maintained in culture medium (10% serum) until harvest. Duplicate cultures of each of the four dose levels of the
test material-exposed cells and of the positive control agent-exposed cells were harvested 18 hours after exposure. 
Two hours prior to harvest, cells were arrested in metaphase by addition of Colcemid.  At harvest, cells were
trypsinized, swollen by hypotonic treatment, fixed on slides and stained with Giemsa.  Mitotic indices were computed by
dividing the number of cells in metaphase by 500 cells examined (per replicate) and expressing this number as a
percentage.  50 cells in metaphase per duplicate (total of 100) at each dose level (including positive controls) were
examined for chromosomal aberrations.  Structural chromosomal abnormalities that were scored included
chromatid and chromosome gaps, chromatid breaks and exchanges, chromosome breaks and exchanges, and chromosomal
disintegration.  Chromatid and chromosome gaps were not included in the number of total aberrations.


Instead of their usual cell line, in this assay Dow used the same strain as NOTOX laboratory in an attempt to more
closely duplicate test conditions.  This change did not produce a positive response as had been found in the 3 NOTOX
lab assays.  A remaining difference between the protocols of the two laboratories was that NOTOX used DMSO to solubilize
DPnB whereas Dow diluted the test material directly into the culture medium without the aid of DMSO.  Because DPnB is
soluble in water up to 5% (i.e., ~50,000 µg/ml), DPnB should be adequately soluble to mix well with the incubation medium
at the concentrations tested such that target cells would be exposed.

The positive control chemicals induced the expected increases in aberration frequencies. In the main assay, the
relative cell survival (RCS) of cultures treated with 5000 ug/ml in the absence and presence of S-9 was 20.9% and
20.8%, respectively. This cytotoxicity was higher than in the preliminary toxicity assay but the next lower dose
evaluated for toxicity (3750 ug/ml), showed no toxicity (note that this dose level was not evaluated for cytogenic
damage).  However, there were no statistically significant increases in the incidence of cells with aberrations in
cultures treated with any of the three concentrations of DPnB, either in the presence or absence of
S-9, as compared to the negative controls.


In conclusion, Dipropylene glycol n-butyl ether did not cause cytotoxicity or chromosomal aberrations under the conditions of this

test. The NOAEL is 5000 µg/ml and no LOAEL was established.


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 µg/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 473 "Genetic Toxicology: In vitro Mammalian
Cytogenetic Test".  The positive control agents gave the expected results showing that the cell line was responsive
to chromosomal aberration insult.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1994-March 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 476
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
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 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 / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April-December 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 475
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Principles of method if other than guideline:
Method: other: No specific protocol guideline cited (e.g., OECD 475: "Mammalian Erythrocyte Micronucleus Test"). General guidelines cited: 40 CFR Part 160, OECD ISBN 92-64-12367-9.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: CD-1 (ICR) BR
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
1% Methocel*
Duration of treatment / exposure:
Single administration
Frequency of treatment:
Single administration
Post exposure period:
Animals were sacrificed at 24 h, 48 h, 72 h post exposure to test material.
Remarks:
Doses / Concentrations:
0, 250, 833, 2500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5 animals per sex per dose per sacrifice time
Control animals:
yes
Positive control(s):
120 mg/kg bw cyclophosphamide
Tissues and cell types examined:
Bone marrow was collected from the femur of each animal.  Cells from the bone marrow were transferred to slides, fixed in methanol, and stained in 5% Giemsa.  One thousand polychromatic erythrocytes were evaluated from each animal and the frequencies of micronucleated polychromatic erythrocytes were recorded.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There were no significant increases in the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in any of the groups treated with the test chemical compared to negative controls at any dose or time point.  The positive control mice showed significant increases in MN-PCE.

Other metabolism studies show that DPnB reaches the bone marrow of mice.  This in vivo assay confirms that that DPnB is not clastogenic to chromosomal material.  See also General Remark above in the In Vitro Genotoxicity section.
Conclusions:
Interpretation of results: negative
Under the experimental conditions used, the test chemical was negative in the mouse bone marrow micronucleus test.
Executive summary:

Groups of CD-1 (ICR) BR mice (5/sex/dose/sacrifice time) were administered single doses (by gavage) of 0, 250, 833,
or 2500 mg DPnB/kg body weight.  The positive control chemical was cyclophosphamide (120 mg/kg).  The negative
control chemical (i.e., the diluent for DPnB) was 1% methocel (10 ml/kg).  Groups of animals were sacrificed by
cervical dislocation at three time intervals: 24, 48, and 72 hours after treatment.  Bone marrow was collected from the
femur of each animal.  Cells from the bone marrow were transferred to slides, fixed in methanol, and stained in 5%
Giemsa.  One thousand polychromatic erythrocytes were evaluated from each animal and the frequencies of
micronucleated polychromatic erythrocytes were recorded.

There were no significant increases in the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in any of
the groups treated with the test chemical compared to negative controls at any dose or time point.  The positive
control mice showed significant increases in MN-PCE.

In conclusion, under the experimental conditions used, the test chemical was negative in the mouse bone marrow micronucleus test.

Other metabolism studies show that DPnB reaches the bone marrow of mice.  This in vivo assay confirms that that DPnB
is not clastogenic to chromosomal material. 

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 2500 mg/kg), positive control agent used, the number of cells scored, and
scoring criteria all followed or exceeded guidance as specified in OECD Guideline 475 "Mammalian Erythrocyte
Micronucleus Test."  The positive control agents gave the expected results showing that the animals were responsive to
clastogenic insult.

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

Additional information

Both in vitro gene mutation studies - in bacteria and in mammalian cells - were clearly negative with and without metabolic activation.

Three out of five in vitro chromosome aberration tests conducted at NOTOX with DPnB in Chinese Hamster Ovary cells, showed positive results. Two in vitro chromosome aberration assays, conducted at Dow Laboratories with Chinese Hamster Ovary cells were negative. In vitro chromosome aberration assays using CHO cells are reported to produce false positive results with certain compounds due to changes in pH or osmolarity of the culture medium. However, in the present studies with DPnB no changes in pH or osmolarity of the culture medium were observed. The presence of peroxides in the samples testing positive also was hypothesized potentially to account for the positive response. Consequently, one of the cytogenicity studies added an antioxidant butylhydroxytoluene (BHT) to reduce the presence of peroxides but BHT (at a concentration low enough to not produce aberrations itself) did not eliminate the clastogenic effects of the sample. Distilling the sample to further remove peroxides also did not eliminate the positive response. The activity of a powerful solvent like DPnB on the cell membrane of sensitive cells could constitute another condition for a false positive. In addition, the discrepancy in results of the in vitro assays at the two laboratories involved could be due to differences in characteristics of the CHO cell lines used, including the sensitivity towards the solvent properties, or to slight differences in the test protocols. Finally, NOTOX used DMSO to solubilize the test material while Dow diluted the test material directly in the culture medium (i.e., did not use DMSO). The combined solvent effects of DMSO and DPnB may have contributed to the positive results in the NOTOX assays.

In an in vivo mouse micronucleus test, DPnB did not induce micronuclei in the bone marrow of mice indicating that DPnB is not clastogenic in vivo. Distribution studies with radiolabeled material show the presence of DPnB in bone marrow.

Based on the equivocal results of the in vitro assays and on the fact that propylene glycol ethers in general are not considered genotoxic, the in vivo mouse bone marrow test becomes definitive in assessing the genotoxic potential of DPnB. Since this in vivo test was negative, it can be concluded that DPnB does not present a genotoxicity hazard.


Short description of key information:
Several in vitro studies according to or equivalent to OECD guidelines 471, 476 and 473 are available for dipropylene glycol butyl ether. In addition, an in vivo study according to OECD guideline 475 is available. All studies (in vitro and in vivo) have been conducted under GLP.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative in vitro gene mutation tests and the negative in vivo micronucleus test, dipropylene glycol butyl ether can be classified as non-genotoxic.