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Diss Factsheets

Administrative data

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:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline 474.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-phenoxypropan-2-ol
EC Number:
212-222-7
EC Name:
1-phenoxypropan-2-ol
Cas Number:
770-35-4
Molecular formula:
C9H12O2
IUPAC Name:
1-phenoxypropan-2-ol
Constituent 2
Reference substance name:
1-phenylpropan-2-ol
EC Number:
211-821-0
EC Name:
1-phenylpropan-2-ol
Cas Number:
698-87-3
IUPAC Name:
1-phenylpropan-2-ol
Details on test material:
Identity: Dowanol-PPh (1-phenoxy-2-hydroxypropane or propylene glycol phenyl ether). CAS # 770-35-4 (also 41593-38-8)
Batch No.: 04114EU
Purity: 93.35 ± 0.02% (by GC/FID area at the 95% confidence level accounting for the level of water (0.2%))
Appearance: Colorless liquid
Source: Aldrich Chemical Company

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8-12 weeks
- Weight at study initiation: not specified
- Assigned to test groups randomly: yes
- Fasting period before study: none
- Housing: The animals were housed 1-2 per cage in stainless steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days prior to start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): standard conditions
- Humidity (%): standard conditions
- Air changes (per hr): standard conditions
- Photoperiod (hrs dark / hrs light): standard conditions

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: [corn oil]
- Justification for choice of solvent/vehicle: recommended by various regulatory agencies
- Concentration of test material in vehicle: not specified
- Amount of vehicle (if gavage): dose volume administered at 10 ml/kg body weight
- Lot/batch no.: not specified
- Purity: not specified
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosing solutions were prepared before dosing on day 1 of study and used for both consecutive days of dose administration. The vehicle used to mix the test material (corn oil) served as the negative control. The concentrations of the test material in the
dosing solutions were verified using liquid chromatography with refractive index detection and external standard quantitation.
Duration of treatment / exposure:
2 days
Frequency of treatment:
single
Post exposure period:
24 hours after the second consecutive adminsitered dose
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg body weight
Basis:
nominal conc.
target concentration - 50 mg/l; observed concentration - 45.1 mg/l
Remarks:
Doses / Concentrations:
1000 mg/kg body weight
Basis:
nominal conc.
target concentration - 100 mg/l; observed concentration - 93.4 mg/l
Remarks:
Doses / Concentrations:
2000 mg/kg body weight
Basis:
nominal conc.
target concentration - 200 mg/l; observed concentration - 182 mg/l
No. of animals per sex per dose:
6 male mice/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): recommended by various regulatory agencies
- Route of administration: oral
- Doses / concentrations: 120 mg/kg

Examinations

Tissues and cell types examined:
The relative body temperatures of the treated animals in the micronucleus test were monitored using programmable transponders (BioMedic Data Systems, Seaford, DE). The temperatures were collected by scanning the microchip immediately prior to each dosing, approximately 2 and 6 hours after each dosing, and prior to sacrifice.
Details of tissue and slide preparation:
Approximately 24 hours after the second consecutive administered dose, all surviving animals were euthanized using central nervous system depression following exposure to carbon dioxide in a closed chamber. Bone marrow samples were obtained from both femurs in the following way. After separating the bone from the adjoining muscles, the distal end of the femur was severed to expose the marrow cavity. A 25-gauge needle was used to aspirate the bone marrow into a 3 ml disposable plastic syringe containing approximate ly 0.5 ml of fetal bovine serum (GIBCO, Grand Island, NY). 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 the serum by gentle aspiration using the syringe and needle. The tubes were centrifuged at 1000 rpm (approximately 80 g) for approximately 5 minutes in a tabletop centrifuge. The supernatant was discarded leaving a small amount of serum covering the pellet. The cell pellet was resuspended using a disposable transfer pipette. Wedge smears were prepared on microscope slides using small portions of the cell suspension. The slides were allowed to air dry and stained with Wright-Giemsa using a Hematek automatic slide stainer.
All slides were coded, scored and decoded upon completion to control for bias. Two 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 smooth contours and varying shapes such as round, almond, or ring (Schmid, 1976). The ratio of PCE to NCE in the bone marrow was determined in the micronucleus test by examining 200 erythrocytes. The ratio was expressed as PCE X 100/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 should be considered nonmutagenic in this test.
Statistics:
The raw data on the counts of MN-PCE for each animal was first transformed by adding 1 to each count and then taking the natural log of the adjusted number. Depending on the results of the sex effect in the range-finding study, the transformed MN-PCE data and the data on percent PCE was analyzed separately by a two-way analysis of variance (when both sexes were used in the study) or a one-way analysis of variance (when only males were used) (Winer, 1971). The sex by dose interaction in the two-way analysis was reviewed and if significant, a one-way analysis of variance was performed for each sex. If different dose levels of the test material were used for each sex, the data was separated by sex prior to statistical analysis. Pairwise comparisons of treated vs. control was done, if the dose effect was significant, by Dunnett’s t-test, one-sided (upper) for MNPCE and two-sided for the percent PCE (Winer 1971). Linear dose-related trend tests was performed if any of the pairwise comparisons yield significant differences. The alpha level at which all the tests were conducted was 0.05.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
ambiguous
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In Phase 1, 1 of 6 males died from treatment in the high dose group (2000 mg/kg/day). Autopsy did not reveal a cause for death. Three males from this group (including the one that died) showed clinical signs of shallow breathing, decreased to absent activity, and hypothermia. The two surviving animals showing hypothermia were placed in a warm environment. No deaths, clinical signs, or hypothermia occurred in the lower dose groups or in the cyclophosphamide control groups. The high dose group showed an average increased frequency of micronuclei. The %MN-PCE (% micronuclei) values from two animals with hypothermia accounted for the increased average of this group and was attributed to hypothermia. These values were 18.0 % and 11.5% while the values in the three other survivors were 1.0%, 4.5%, and 3.0%, similar to the corn oil control group values. Animals treated with lower doses of Propylene glycol phenyl ether showed no effects on any parameter.
In Phase 2, the effects seen in Phase 1 were observed again in the 2000 mg/kg/day group. Although not statistically significant, the %MN-PCE was elevated once more. Marked hypothermia was observed yet again at this dose level only in both sexes. As in Phase 1, the ratio of polychromatic (PCE) to normo-chromatic erythrocytes (NCE) was decreased in the high dose group. Body weights were unaffected in either Phase.

Any other information on results incl. tables

none

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The statistically identified increase in MNPCE frequency at the 2000 mg/kg dose level is interpreted to be the result of treatment-induced hypothermia and not due to a direct action of phenoxypropanol and/or its metabolites on cellular targets responsible for micronucleus induction. Hence, the test material is not considered to be a clastogenic agent.
Executive summary:

In this study, groups of 6 male mice (outbred CD-1 (1CR)BR, 8-12 weeks old) per dose level from Charles River Laboratories, Portage, MI were administered 0, 500, 1000, or 2000 mg PPh/kg body weight by gavage on 2 consecutive days by oral intubation. PPh was mixed in corn oil to achieve a constant dosing volume among groups. These doses were selected from a pilot dose-range finding study. Because hypothermia resulted from treatment in this Phase 1 study, particularly in the high dose subjects, the experiment was repeated with both sexes (Phase 2) with 6 additional animals in the high dose group to serve as replacements in the event of mortality.

For PPh, dosing solution concentrations were adjusted (diluted in corn oil) in order to provide dosing volume of 2 ml/kg body weight. Cyclophosphamide monohydrate was used as the positive control agent and was administered in distilled water at a dose level of 120 mg/kg body weight. Mice were observed for mortality and clinical signs of toxicity at least once/day following the initial dose. Body temperature was collected using an implanted transponder; temperatures were recorded immediately prior to dosing, 6-hours post-dosing, and prior to termination. 24-Hours after the last dose, mice were euthanized with CO2 and bone marrow was collected by aspiration from both femurs. For each subject, 2000 polychromatic erythrocytes (PCEs) were examined microscopically for the presence of micronuclei (MN-PCE). The number of MN-PCE was expressed as a percentage of total PCE. In Phase 1, 1 of 6 males died from treatment in the high dose group (2000 mg/kg/day). Autopsy did not reveal a cause for death. Three males from this group (including the one that died) showed clinical signs of shallow breathing, decreased to absent activity, and hypothermia. The two surviving animals showing hypothermia were placed in a warm environment. No deaths, clinical signs, or hypothermia occurred in the lower dose groups or in the cyclophosphamide control groups. The high dose group showed an average increased frequency of micronuclei. The %MN-PCE (% micronuclei) values from two animals with hypothermia accounted for the increased average of this group and the authors of the study attributed the increase to hypothermia.

These values were 18.0 % and 11.5% while the values in the three other survivors were 1.0%, 4.5%, and 3.0%, similar to the corn oil control group values.

In Phase 2, the effects seen in Phase 1 were observed again in the 2000 mg/kg/day group. Although not statistically significant, the %MN-PCE was elevated once more. Marked hypothermia was observed yet again at this dose level only in both sexes. As in Phase 1, the ratio of polychromatic (PCE) to normo-chromatic erythrocytes (NCE) was decreased in the high dose group. Body weights were unaffected in either Phase. Only males (6/dose level) were used in phase 1 while male and females (6/sex/dose level) were used in phase two. The authors of this study concluded that, most likely, the increased incidence of micronuclei seen at 2000 mg/kg/day was attributable to the hypothermia induced by PPh and not as a direct clastogenic effect from PPh. The authors cited papers by Asanami et al. (Asanami, S., Shimono, K., (1997). High body temperature induces micronuclei in mouse bone marrow. Mutation Research, 390:70-83 and Asanami, S., Shimono, K., Kaneda, S., (1998). Transient hypothermia induces micronuclei in mice. Mutation Research, 413:7-14) showing that agents such as reserpine and chlorpromazine, which induce hypothermia, cause increased micronuclei as an indirect result of this physiological change. Asanami et al. hypothesize that hypothermia may cause clastogenic injury by interfering with microtubule assembly and spindle function. Since a separate, additional group at the high dose level was not placed in a warmed environment after treatment to directly test the hypothesis of hypothermia causing the increased micronuclei, the possibility that the increased incidence of micronuclei at the high dose was directly attributable to PPh cannot be excluded. On the other hand, it is relevant to note that the next lower dose (still a very large dose of 1000 mg/kg) did not cause hypothermia or an increase in micronuclei. If the increase was directly attributable to PPh and not hypothermia, it is significant that only a marginal effect resulted (not statistically significant when repeated in a second experiment), which required a very large dose of 2000 mg/kg

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