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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1990 - March 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study equivalent to OECD guideline 412
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:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
Specific protocol guideline not specified. Followed requirements of OECD Guideline 412 "Repeated Dose Inhalation Toxicity: 28-day or 14-day Study"
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Dowanol-DPnB (n-butoxypropoxypropanol
or dipropylene glycol normal-butylether).
CAS # 29911-28-2
Batch No.: 891115
Purity: see below
Supplied as: Not reported.
Appearance: Colorless liquid.
Administered as: Aerosol
Vapor pressure: 0.06 mmHg at 25°C (79 ppm at 1 atm)
Specific Gravity: 0.91 g/ml.
Solubility: 5% in water.
Stability: Stable up to 200°C.

Dipropylene glycol n-butyl ether isomers: 99.70%
Dipropylene glycol: 0.17%

Dipropylene glycol n-butyl ether (DPnB) is a mixture of 4
possible isomers with the major isomers being
1-(1-n-butoxy-2-propoxy)-2-propanol and
2-(1-n-butoxy-2-propoxy)-1-propanol.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Age at dosing:         6 weeks of age.
Source:                Charles River Breeding Laboratory, Kingston, N.Y.
Acclimation period:    At least one week.
Weight range (start of study):     Males:  166 to 188 grams;  Females: 110 to 122 grams.
Assignment to groups:  Computerized, weight-stratification and random number-based procedure.
Diet:                  Purina Certified Rodent Chow #5002 (Purina Mills, Inc., Richmond, ID).
Access to food:        Available ad libitum except during exposures.
Access to food:        Available ad libitum except during exposures.
Method of  Identification:        Ear tags.
Housing:               Individually during non-exposure periods (type housing not specified). In polycarbonate tubes during daily 6-hr exposures.  Animals were acclimated to the tubes 2 days prior to exposure for 2 to 4 hrs.

Environmental Conditions (for non-exposure periods):
Temperature:           Not specified.
Humidity:              Not specified.
Air changes:           Not specified.
Photoperiod:           12 hr light/12 hr dark.

Environmental Conditions (for exposure periods):
Temperature:           22.5 - 24.5°C (recorded at the end of each exposure period).
Humidity:              34-60% (recorded at the end of each exposure period).
Airflow:               30 liters/min.
Air changes:           >25 air changes per hour.
Photoperiod:           12 hr light/12 hr dark.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: MMAD and GSD are not reported since the particle sizes were not log-normally distributed.
Details on inhalation exposure:
Groups of 5 male and 5 female young adult Fischer 344 rats were exposed to an aerosol atmosphere of DPnB, at concentrations of 0, 200, 810, or 2010 mg/m3 (0, 25, 100, or 250 ppm), by nose-only exposure, 6 hr/day, 5 d/wk over a 2 week period for a total of 9 exposures.  

Polycarbonate tubes containing the subjects (nose cones) were attached to a 42-liter ADG nose-only conical inhalation chamber (30 x 60 cm) with an airflow of 30 liters/min. Aerosol was generated by metering DPnB into a stainless steel ¼ J spray nozzle using a FMI pump.  DPnB was mixed with compressed air in the spray nozzle and test material was sprayed into the chamber as an aerosol.  Aerosol total mass concentrations were measured gravimetrically on pre-weighed Teflon (TE36) filters (0.45 micron pore size) at least three times per day for each chamber.  Aerodynamic particle size was characterized 3 times (per exposure period or once for the study not specified) for each chamber using a 6-stage cascade impactor with increasingly diminishing pore sizes in the 6 stages.  Temperature and humidity were monitored at the end of each 6-hour exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The mass concentration of aerosol present in the chamber was determined gravimetrically on preweighed teflon (TE36) filters (0.45 u pore size, Schleicher & Schuell, Inc., Keene, NH) at least 3 times per day for each chamber (except control) by drawing samples from a vertical stainless steel tube which projected into the animal breathing zone. The analytical exposure concentrations were calculated from these determinations using a time-weighted-average (TWA) method.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 h/d, 5 d/w (9 exposures)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.20, 0.81, 2.01 mg/l (0, 200, 810, 2010 mg/m3)
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, sham-exposed
Details on study design:
Post-exposure period: none
Positive control:
none

Examinations

Observations and examinations performed and frequency:
Rats were observed after each exposure for mortality and clinical signs of toxicity.  The subjects were weighed on days 1, 3, 5, 8, and 11 of the study.Ophthalmic examination was conducted prior to the first exposure and at sacrifice.  Hematology, clinical chemistry, and urinalyses were conducted prior to sacrifice.  
Sacrifice and pathology:
All animals were subjected to gross necropsy, major organs were weighed, and over 50 tissues were collected and processed into slides for histological examination. 
Other examinations:
Rats were observed after each exposure for mortality and clinical signs of toxicity.  The subjects were weighed on days 1, 3, 5, 8, and 11 of the study.  Ophthalmic examination was conducted prior to the first exposure and at sacrifice.  Hematology, clinical chemistry, and urinalyses were conducted prior to sacrifice.  All animals were subjected to gross necropsy, major organs were weighed, and over 50 tissues were collected and processed into slides for histological examination. 
Statistics:
Chamber concentration, temperature, relative humidity, airflow and differential WBC count were reported with descriptive statistics only (mean and standard deviation). All remaining parameters examined statistically were first tested for equality of variance using Bartlett's test. If the results from Bartlett's test reject the equality of variances, the parameter was flagged for careful evaluation of results. Otherwise all parameters were subjected to appropriate parametric analysis. In-life body weights were evaluated using a 3-way analysis of variance (ANOVA) with the factors of sex, dose and time interval 9Winer, 1971). Organ weights (absolute and relative except testes), terminal body weights, hematologies, clinical chemistries and urinalysis (specific gravity) were evaluated using a 2-way (ANOVA) with the factors of sex and dose (Winer, 1971). Results for absolute and relative testes weights were analyzed using a one way ANOVA. If significant dose effects were determined in the one-way ANOVA, then separate doses were compared to controls using separate one-way ANOVA's for each dose compared to control; a Bonferroni correction was used.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
Survival:  All rats survived the nine exposures over the 14-day study period. 

Clinical signs:  Some rats were soiled with urine and feces from being in the nose cones after exposures.  This was attributed by the authors to the stress of confinement in the polycarbonate tubes.  All males and some female rats in the high exposure group exhibited lethargic behavior.  This behavior disappeared in most subjects after the second exposure (1 male was lethargic on test day 9) and was not evident in the low or mid-exposure groups.

Body weights:  Males from the high exposure group lost significantly more body weight than controls or lower exposure groups, indicating a treatment related effect.  The control, low, and mid-exposure groups lost weight during the initial phase of exposure but body weights in these groups rebounded and exceeded initial body weights by the end of the study.  Male body weights from the high exposure group were statistically different from controls and were still lower than their initial weights at the end of the study.  Females in all groups showed an initial body weight loss due to confinement stress in the polycarbonate tubes but no treatment related effects were evident (i.e., all paralleled air-only controls).

Ophthalmological Examination:  Although some eye lesions were found, no effects attributable to treatment were noted.  Specifically, two females in the high exposure group were found to have bilateral cornel opacities (total involvement) but these were not attributed to DPnB by the study authors.

Gross pathology:  A few grossly observable pathological lesions were noted at necropsy but were judged to be spontaneous or incidental to the stressful regimen of nose-only treatment and not related to DPnB exposure.

Organ weights:  Absolute and relative liver weights in both sexes from the mid and high exposure groups were statistically increased above controls.  Liver weight changes were accompanied by increased hepatocyte size but little histopathology was evident and therefore liver weight increases were considered adaptive (e.g., due to MFO induction) rather than a toxicological response to DPnB exposure.  Relative kidney weights were increased in high dose males, which the authors conjectured might reflect lower body weights.  No differential response existed between control and high exposure male kidney histopathology.  Both sexes from the high exposure group exhibited statistically decreased absolute brain weights but males from this group also showed a significant relative brain weight increase.  Relative lung weights in both sexes were statistically increased (absolute lung weights only in the mid-exposure males also were statistically increased). Statistically significant large decreases in thymus (males only; absolute and relative) and spleen weights (males only; absolute only) were also observed in the high exposure group.  The authors considered organ weight changes to be related to body weight decreases and therefore secondary to the stress-related influence from confinement in the polycarbonate exposure tubes.  This conclusion was supported by a lack of accompanying histopathology or correlating clinical toxicity in most of these organs.

Hematology:  Both sexes in the high-exposure group showed a statistical decrease in RBCs, hematocrits, and hemoglobin when compared to controls.  These findings were not considered treatment related because 1) decreases were slight, 2) values were within laboratory historical control ranges (for HGB and HCT) or nearly so (RBC), 3) erythropoiesis was not evident when slides were examined of bone marrow, and 4) no evidence of hemolysis was present.

Clinical Chemistry:  Urea nitrogen was statistically increased in high-exposure males and total protein was statistically decreased in both sexes from all DPnB-exposure groups.  Other parameters that were statistically different from controls were considered spurious because they did not follow a dose-response or were only slightly different from controls.  These included alterations in ALT, albumin, globulin, cholesterol, potassium, and calcium.
Urinalysis:  No changes were observed in any urinalysis parameters. Histopathology:  In the high-exposure group, 4 males and 1 female exhibited increased hepatocyte size across the liver lobule with a suggestion of accompanying damage (e.g., slight vacuolation or multifocal necrosis).  

Histological damage to this organ was considered by the authors to be related to compressive trauma during the treatment periods.  Increased hepatocyte size also was noted in two males from the mid-exposure group.  In the anterior nasal cavity, rats from the mid and high-exposure groups exhibited 1) multifocal epithelial hyperplasia (1 female at the mid-dose; 4 males and 3 females at the high dose) and 2) squamous metaplasia (1 male and 4 females at the mid-dose; 5 males and females at the high dose).  Nasal effects were considered a direct response to irritation from DPnB typical for mucosal tissue and were sometimes accompanied by suppurative inflammation or degeneration of the olfactory epithelium.  No adverse effects were noted in the deeper respiratory tract.  Slight to moderate lymphoid depletion in the thymus and spleen were noted in some rats (primarily males) in the mid and high exposure groups.  No evidence was present for a hemolytic effect in these or other organs/tissues and the lymphoid effect was considered secondary to weight loss in the two highest exposure groups. Characterization of the aerosol atmosphere:  Nominal concentrations were approximately twice actual concentrations for the 200 and 810 mg/m3 exposure levels. Nominals agreed with actual at the 2010 mg/m3 level.  Mass median aerodynamic diameter and the geometric standard deviation were not calculated because the aerosol particle size was not log-normally distributed.  The percentage of particles under 3 microns (i.e., deep lung respirable) were 49%, 46%, and 62% for the 200, 810, and 2010 mg/m3 exposure levels, respectively.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
200 mg/m³ air (analytical)
Sex:
male/female
Basis for effect level:
other: liver weight increase and nasal irritation
Dose descriptor:
LOAEL
Effect level:
0.81 other: mg/l
Sex:
male/female
Basis for effect level:
other: effects on the liver and nasal mucosa

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
All rats exposed to aerosols of DPnB at concentrations of 0, 200, 810, or 2010 mg/m3 survived a total of nine 6-hour exposures over a period of two weeks with minimal clinical effects (lethargy for the first few days). The NOAEL for DPnB in this study was 200 mg/m3 and the LOAEL was 810 mg/m3 based on effects on the liver and nasal mucosa.
Executive summary:

Groups of 5 male and 5 female young adult Fischer 344 rats were exposed to an aerosol atmosphere of DPnB, at
concentrations of 0, 200, 810, or 2010 mg/m3 (0, 25, 100, or 250 ppm), by nose-only exposure, 6 hr/day, 5 d/wk over a 2
week period for a total of 9 exposures.  Rats were observed after each exposure for mortality and clinical signs of
toxicity.  The subjects were weighed on days 1, 3, 5, 8, and 11 of the study.  Ophthalmic examination was conducted prior
to the first exposure and at sacrifice.  Hematology, clinical chemistry, and urinalyses were conducted prior to
sacrifice.  All animals were subjected to gross necropsy, major organs were weighed, and over 50 tissues were
collected and processed into slides for histological examination. 

Polycarbonate tubes containing the subjects (nose cones) were attached to a 42-liter ADG nose-only conical inhalation
chamber (30 x 60 cm) with an airflow of 30 liters/min. Aerosol was generated by metering DPnB into a stainless
steel ¼ J spray nozzle using a FMI pump.  DPnB was mixed with compressed air in the spray nozzle and test material
was sprayed into the chamber as an aerosol.  Aerosol total mass concentrations were measured gravimetrically on
pre-weighed Teflon (TE36) filters (0.45 micron pore size) at least three times per day for each chamber.  Aerodynamic
particle size was characterized 3 times (per exposure period or once for the study not specified) for each chamber using
a 6-stage cascade impactor with increasingly diminishing pore sizes in the 6 stages.  Temperature and humidity were
monitored at the end of each 6-hour exposure.

Survival:  All rats survived the nine exposures over the 14-day study period. 

Clinical signs:  Some rats were soiled with urine and feces from being in the nose cones after exposures.  This was
attributed by the authors to the stress of confinement in the polycarbonate tubes.  All males and some female rats in
the high exposure group exhibited lethargic behavior.  This behavior disappeared in most subjects after the second
exposure (1 male was lethargic on test day 9) and was not evident in the low or mid-exposure groups.

Body weights:  Males from the high exposure group lost significantly more body weight than controls or lower
exposure groups, indicating a treatment related effect.  The control, low, and mid-exposure groups lost weight during the
initial phase of exposure but body weights in these groups rebounded and exceeded initial body weights by the end of
the study.  Male body weights from the high exposure group were statistically different from controls and were still
lower than their initial weights at the end of the study.  Females in all groups showed an initial body weight loss due
to confinement stress in the polycarbonate tubes but no treatment related effects were evident (i.e., all paralleled
air-only controls).

Ophthalmological Examination:  Although some eye lesions were found, no effects attributable to treatment were noted.
 Specifically, two females in the high exposure group were found to have bilateral cornel opacities (total involvement)
but these were not attributed to DPnB by the study authors.

Gross pathology:  A few grossly observable pathological lesions were noted at necropsy but were judged to be
spontaneous or incidental to the stressful regimen of nose-only treatment and not related to DPnB exposure.

Organ weights:  Absolute and relative liver weights in both sexes from the mid and high exposure groups were
statistically increased above controls.  Liver weight changes were accompanied by increased hepatocyte size but
little histopathology was evident and therefore liver weight increases were considered adaptive (e.g., due to MFO
induction) rather than a toxicological response to DPnB exposure.  Relative kidney weights were increased in high
dose males, which the authors conjectured might reflect lower body weights.  No differential response existed
between control and high exposure male kidney histopathology.  Both sexes from the high exposure group
exhibited statistically decreased absolute brain weights but males from this group also showed a significant relative
brain weight increase.  Relative lung weights in both sexes were statistically increased (absolute lung weights only in
the mid-exposure males also were statistically increased). Statistically significant large decreases in thymus (males
only; absolute and relative) and spleen weights (males only; absolute only) were also observed in the high exposure
group.  The authors considered organ weight changes to be related to body weight decreases and therefore secondary to
the stress-related influence from confinement in the polycarbonate exposure tubes.  This conclusion was supported
by a lack of accompanying histopathology or correlating clinical toxicity in most of these organs.

Hematology:  Both sexes in the high-exposure group showed a statistical decrease in RBCs, hematocrits, and hemoglobin
when compared to controls.  These findings were not considered treatment related because 1) decreases were
slight, 2) values were within laboratory historical control ranges (for HGB and HCT) or nearly so (RBC), 3)
erythropoiesis was not evident when slides were examined of bone marrow, and 4) no evidence of hemolysis was present.

Clinical Chemistry:  Urea nitrogen was statistically increased in high-exposure males and total protein was
statistically decreased in both sexes from all DPnB-exposure groups.  Other parameters that were statistically different
from controls were considered spurious because they did not follow a dose-response or were only slightly different from
controls.  These included alterations in ALT, albumin, globulin, cholesterol, potassium, and calcium.

Urinalysis:  No changes were observed in any urinalysis parameters.

Histopathology:  In the high-exposure group, 4 males and 1 female exhibited increased hepatocyte size across the liver
lobule with a suggestion of accompanying damage (e.g., slight vacuolation or multifocal necrosis).  Histological
damage to this organ was considered by the authors to be related to compressive trauma during the treatment periods. 
Increased hepatocyte size also was noted in two males from the mid-exposure group.  In the anterior nasal cavity, rats
from the mid and high-exposure groups exhibited 1) multifocal epithelial hyperplasia (1 female at the mid-dose;
4 males and 3 females at the high dose) and 2) squamous metaplasia (1 male and 4 females at the mid-dose; 5 males
and females at the high dose).  Nasal effects were considered a direct response to irritation from DPnB typical
for mucosal tissue and were sometimes accompanied by suppurative inflammation or degeneration of the olfactory
epithelium.  No adverse effects were noted in the deeper respiratory tract.  Slight to moderate lymphoid depletion in
the thymus and spleen were noted in some rats (primarily males) in the mid and high exposure groups.  No evidence was
present for a hemolytic effect in these or other organs/tissues and the lymphoid effect was considered secondary to weight loss in the two highest exposure groups.

Characterization of the aerosol atmosphere:  Nominal concentrations were approximately twice actual concentrations for the 200 and 810 mg/m3 exposure levels. Nominals agreed with actual at the 2010 mg/m3 level.  Mass
median aerodynamic diameter and the geometric standard deviation were not calculated because the aerosol particle
size was not log-normally distributed.  The percentage of particles under 3 microns (i.e., deep lung respirable) were
49%, 46%, and 62% for the 200, 810, and 2010 mg/m3 exposure levels, respectively.

This study tested the effects of DPnB at concentrations much higher than the previous 2-week inhalation study (high
exposure level 40 ppm).  A concentration of 2010 mg/m3 is equivalent (if converted to a vapor concentration) of
approximately 260 ppm.  Units of ppm have not been used because a vapor concentration this high could not be
generated with DPnB due to its low vapor pressure.

In conclusion, all rats exposed to aerosols of DPnB at concentrations of 0, 200, 810, or 2010 mg/m3 survived a total of nine 6-hour exposures over a period of two weeks with minimal clinical effects (lethargy for the first few days). The primary effects from DPnB exposure were decreased body weights in rats of both sexes at 2010 mg/m3 and histopathological lesions in the liver and nasal cavities in both sexes at 810 and 2010 mg/m3. The stress of the 6-hour confinement in the polycarbonate exposure tube contributed to the body weight decreases. The liver changes, although accompanied by slight necrosis in some instances, were characterized primarily by increased hepatocyte size, suggesting an adaptive response (i.e., mixed function oxidase enzyme induction). The observed liver weight increases support this conclusion. Hyperplasia, metaplasia, degeneration, and/or inflammation of the anterior nasal mucosa were considered a direct response to the irritant properties of DPnB, typical in mucous membranes. Depletion of cells in the thymus and spleen were considered secondary to the stress of confinement in polycarbonate tubes for the nine 6-hour exposure periods. The NOAEL for DPnB in this study was 200 mg/m3 and the LOAEL was 810 mg/m3 based on effects on the liver and nasal mucosa.

This study was identified as key for this toxicity endpoint because of the methods followed (which were comprehensively
documented in the report).  The report included GLP and Quality Assurance statements, signed by the Study Director
and Head of the QA Unit, respectively.  Although the study report did not specify that OECD Guideline 412 "Repeated
Dose Inhalation Toxicity: 28-day or 14-day Study" was followed, the study satisfied the methods stipulated in this
protocol.  Specifically, the numbers and type of test animals used and their husbandry conditions were as
prescribed in the guidance.  Test material characterization was adequate.  The dose level tested satisfied the
appropriate OECD upper limit (i.e., the maximum practically attainable), the length of the observation period (14 days)
was sufficient, and the toxicity endpoints monitored were typical for this type assay and adequately recorded.