Registration Dossier

Administrative data

Description of key information

For repeated dose toxicity of DPnB, studies via oral, dermal and inhalation exposure are availalbe.
Oral: 14-d oral gavage and 90-d dietary studies in rats. These are GLP-studies conducted according to OECD guidelines 407&408.
Dermal: 90-d dermal study in rats. This is a GLP-study conducted according to OECD guideline 411.
Inhalation: one 14-d vapor inhalation study in rats and one 14-d aerosol inhalation study in rats. Both are GLP-studies conducted according to OECD guideline 412.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
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 408
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
yes
Species:
rat
Strain:
other: SSprague-Dawley, outbred SPF - Stock KFM: SPRD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: KPF - SPRD
- Diet: ad libitum
- Water: ad libitum
- Age at study initiation: 10 weeks
- Weight at study initiation: 222-265 g for males, 172-207 for females
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + or - 3
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12
- Air change: 10-15 change/hour

For details, pls. see table 1 below
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Four groups of Sprague-Dawley rats (20/sex/dose level) received dipropylene glycol n-butyl ether (DPnB) in their feed at concentrations equivalent to target doses of 0, 200, 450, or 1000 mg/kg-day for 13 weeks.  Five additional rats per sex were added to each group.  These additional rats received DPnB in their feed for only four weeks and then were sacrificed in order to assess DPnB toxicity at this interim period.  
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were sent to analytical laboratory of RCC for analysis of homogenicity and concentration.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0, 200, 450, or 1000 mg/kg-day
Basis:
nominal in diet
No. of animals per sex per dose:
20/sex/dose
Control animals:
yes
Details on study design:
Four groups of Sprague-Dawley rats (20/sex/dose level) received dipropylene glycol n-butyl ether (DPnB) in their feed at concentrations equivalent to target doses of 0, 200, 450, or 1000 mg/kg-day for 13 weeks.  Five additional rats per sex were added to each group.  These additional rats received DPnB in their feed for only four weeks and then were sacrificed in order to assess DPnB toxicity at this interim period.  

Nominal doses and weekly ranges of doses are reported in the table below.  Concentrations of DPnB in feed were adjusted on a weekly basis, based on food consumption patterns, to achieve the desired dose.  For males, concentrations of DPnB in feed ranged from: 1) 2200-3400 ppm for the 200 mg/kg-d group, 2) 4950-7200 ppm for the 450 mg/kg-d group, and 3) 11000-16000 ppm for the 1000 mg/kg-d group.  For females, concentrations of DPnB in feed ranged from: 1) 2150-2700 ppm for the 200 mg/kg-d group, 2) 4840-6000 ppm for the 450 mg/kg-d group, and 3) 10750-13200 ppm for the 1000 mg/kg-d group.

Study design: see table 2 below
Positive control:
none
Observations and examinations performed and frequency:
Rats were observed for mortality twice daily and for clinical signs of toxicity once per day.  Once weekly, animals were given a more detailed clinical examination with palpation for masses.  Body weights, water, and food consumption were monitored weekly.  Ophthalmological examinations were conducted prior to treatment and at sacrifice (interim animals included).  Hematology, clinical chemistry, and urinalysis evaluations were conducted at 4 and 13 weeks.  
Sacrifice and pathology:
At sacrifice, all control and high dose animals were subjected to complete necropsy and histopathological evaluations.  Gross lesions were recorded at necropsy.  Selected organs were weighed and over 40 tissues were collected from all animals and fixed for histopathological evaluation.  Only tissues from control and high-dose animals were evaluated histopathologically.
Other examinations:
none
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):
no effects observed
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:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Absolute and relative liver weights were increased in high dose males.   In females at the high dose level, absolute and relative kidney weights were increased with no accompanying histopathology.  Slight alterations in clinical chemistries, electrolytes, and hematology also were noted in both sexes at the high dose level.  No changes in any other monitored parameters were noted at any dose level.  The NOAEL for DPnB is 450 mg/kg-day and the LOAEL is 1000 mg/kg-day (for organ weight changes).

Morbidity/Mortality:  All rats survived treatment with the test compound.

Clinical signs:  No treatment-related signs reported.

Food Consumption:  A slight increase in relative food consumption (to body weight) but not absolute food consumption was noted in high dose males.

Water Consumption:  No effect on water consumption was noted.

Body weights:  Body weights in high dose males were slightly decreased during the first three weeks of treatment.  Although not statistically significant thereafter, this trend continued in the high dose males throughout the study. Body weights were unaffected in high dose females or in either sex from lower treatment groups.  

Organ Weights:  High-dose males showed slightly increased liver weight to body weight ratios.  Absolute liver weights and liver weight to brain weight ratios were not statistically different from controls.

Clinical Chemistries:  The following parameters were statistically altered from controls, in one or both sexes, most often in the high dose group.  The alterations are slight in nature but are consistent with liver toxicity, although histopathology did not confirm damage to this organ.  Urea:  Slightly elevated in mid and high dose males and females at 4 and 13 weeks.  Cholesterol:  Slightly elevated in high dose male and females at 4 weeks and females at 13 weeks.  Gamma-glutamyl transferase:  Slightly elevated in high dose males at 4 and 13 weeks.  Glucose:  Slightly elevated In high dose females at 4 and 13 weeks.  Potassium:  Slightly elevated in high dose males and females at 4 and 13 weeks.

Hematology:  No treatment-related changes noted. Urinalysis:  High dose males were the only subjects that showed effects possibly related to treatment.  When compared to negative controls, urine of high dose males showed: slightly lower urinary pH (4 & 13 weeks), moderately increased numbers of transitional epithelial cells (4 weeks), slightly to moderately decreased sodium excretion (4 & 13 weeks), and moderately increased magnesium excretion (4 & 13 weeks).  Other changes occurred but were considered unrelated to treatment.

Ophthalmological Examinations:  No treatment-related lesions noted. Macroscopic Examinations:  No treatment-related lesions noted.

Histological Examinations:  No lesions reported except for "a coarse yellow-brown pigment . . .noted in one female rat from group 4.  This pigment was located mainly in hepatocytes of zone 1 and occasionally in Kupffer cells.  The identity of this pigment was not established."
Dose descriptor:
NOAEL
Effect level:
450 other: mg/kg
Sex:
male/female
Basis for effect level:
other: increase in kidney and liver weights without accompanying histopathology
Critical effects observed:
not specified
Conclusions:
Body weights were decreased slightly but statistically in high-dose males (1000 mg/kg-d). Livers were enlarged but without accompanying histopathology in high-dose males. Liver findings were corroborated by clinical chemistry results in which some parameters reflective of liver injury
were slightly elevated in the high dose groups of either or both sexes (urea was elevated in mid-dose subjects but did not exhibit a dose-response). Some urinary parameters in high dose rats were altered (only in high-dose subjects). Most of these findings occurred after 4 as well as after 13 weeks of exposure to DPnB. The NOAEL is 450 mg/kg-day and the LOAEL, based on decreased body weights, increased liver weights (without histopathology) and slight alterations in clinical chemistry parameters, is 1000 mg/kg-d.
Executive summary:

Four groups of Sprague-Dawley rats (20/sex/dose level) received dipropylene glycol n-butyl ether (DPnB) in their feed at concentrations equivalent to target doses of 0, 200, 450, or 1000 mg/kg-day for 13 weeks.  Five additional rats per sex were added to each group.  These additional rats received DPnB in their feed for only four weeks and then
were sacrificed in order to assess DPnB toxicity at this interim period.  Nominal doses and weekly ranges of doses
are reported in the table below.  Concentrations of DPnB in feed were adjusted on a weekly basis, based on food
consumption patterns, to achieve the desired dose.  For males, concentrations of DPnB in feed ranged from: 1) 2200-3400 ppm for the 200 mg/kg-d group, 2) 4950-7200 ppm for the 450 mg/kg-d group, and 3) 11000-16000 ppm for the
1000 mg/kg-d group.  For females, concentrations of DPnB in feed ranged from: 1) 2150-2700 ppm for the 200 mg/kg-d
group, 2) 4840-6000 ppm for the 450 mg/kg-d group, and 3) 10750-13200 ppm for the 1000 mg/kg-d group.

Rats were observed for mortality twice daily and for clinical signs of toxicity once per day.  Once weekly,
animals were given a more detailed clinical examination with palpation for masses.  Body weights, water, and food
consumption were monitored weekly.  Ophthalmological examinations were conducted prior to treatment and at
sacrifice (interim animals included).  Hematology, clinical chemistry, and urinalysis evaluations were conducted at 4
and 13 weeks.  At sacrifice, all control and high dose animals were subjected to complete necropsy and
histopathological evaluations.  Gross lesions were recorded at necropsy.  Selected organs were weighed and over 40
tissues were collected from all animals and fixed for histopathological evaluation.  Only tissues from control and
high-dose animals were evaluated histopathologically.

Absolute and relative liver weights were increased in high dose males.   In females at the high dose level, absolute
and relative kidney weights were increased with no accompanying histopathology.  Slight alterations in clinical
chemistries, electrolytes, and hematology also were noted in both sexes at the high dose level.  No changes in any other
monitored parameters were noted at any dose level.  The NOAEL for DPnB is 450 mg/kg-day and the LOAEL is 1000
mg/kg-day (for organ weight changes).

Morbidity/Mortality:  All rats survived treatment with the test compound.

Clinical signs:  No treatment-related signs reported.

Food Consumption:  A slight increase in relative food consumption (to body weight) but not absolute food consumption was noted in high dose males.

Water Consumption:  No effect on water consumption was noted.

Body weights:  Body weights in high dose males were slightly decreased during the first three weeks of treatment. 
Although not statistically significant thereafter, this trend continued in the high dose males throughout the study.
 Body weights were unaffected in high dose females or in either sex from lower treatment groups.
 
Organ Weights:  High-dose males showed slightly increased liver weight to body weight ratios.  Absolute liver weights
and liver weight to brain weight ratios were not statistically different from controls.

Clinical Chemistries:  The following parameters were statistically altered from controls, in one or both sexes,
most often in the high dose group.  The alterations are slight in nature but are consistent with liver toxicity,
although histopathology did not confirm damage to this organ.  Urea:  Slightly elevated in mid and high dose males
and females at 4 and 13 weeks.  Cholesterol:  Slightly elevated in high dose male and females at 4 weeks and
females at 13 weeks.  Gamma-glutamyl transferase:  Slightly elevated in high dose males at 4 and 13 weeks.  Glucose: 
Slightly elevated In high dose females at 4 and 13 weeks. 

Potassium:  Slightly elevated in high dose males and females at 4 and 13 weeks.

Hematology:  No treatment-related changes noted.

Urinalysis:  High dose males were the only subjects that showed effects possibly related to treatment.  When compared
to negative controls, urine of high dose males showed: slightly lower urinary pH (4 & 13 weeks), moderately
increased numbers of transitional epithelial cells (4 weeks), slightly to moderately decreased sodium excretion (4
& 13 weeks), and moderately increased magnesium excretion (4 & 13 weeks).  Other changes occurred but were considered
unrelated to treatment.

Ophthalmological Examinations:  No treatment-related lesions noted.

Macroscopic Examinations:  No treatment-related lesions noted.

Histological Examinations:  No lesions reported except for "a coarse yellow-brown pigment . . .noted in one female rat
from group 4.  This pigment was located mainly in hepatocytes of zone 1 and occasionally in Kupffer cells. 
The identity of this pigment was not established."

The results from this study indicate low toxicity for DPnB. 
No evidence was found for hemolytic activity.

The NOAEL is 450 mg/kg-day and the LOAEL is 1000 mg/kg-d, based on decreased body weights, increased liver weights (without histopathology) and slight alterations in clinical chemistry parameters.

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.  The study and report followed OECD Protocol 408: "Repeated Dose 90-day Oral
Toxicity Study in Rodents."  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 of test material complied with
guidance, the length of the treatment period (90 days) was sufficient, and evaluation criteria and statistical methods
were typical for this type assay and adequately recorded.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
450 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
good

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
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.
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)
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
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.
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.
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
Critical effects observed:
not specified
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.

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July -December 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study equivalent to OECD guideline 412
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
Age at dosing:         At least 5 weeks of age.
Source:                Charles River Breeding Laboratory,Kingston, N.Y.
Acclimation period:    At least one week.
Animals were acclimated to the tubes 4 days prior to exposure (1 hr/ on day -4, 3 hr on day -3, and  6 hrs -2 and -1).
Weight range  (start of study):      Males: 206 to 224 grams;  Females: 139 to 152 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 in glass jars.
Access to water:       Available ad libitum (glass bottles).
Method of  Identification:        Ear tags.
Housing:               Individually in stainless steel cages with wire-mesh bottoms during non-exposure periods.
In poly-carbonate tubes during daily 6-hr exposures.  


Environmental Conditions (for nonexposure periods):
Temperature:           Approximately 22°C (recorded at the end of each exposure period).   
Humidity:              50% (recorded at the end of each exposure period).
Air changes:           not specified.
Photoperiod:           12 hr light/12 hr dark.
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: n/a
Details on inhalation exposure:
In a 2-week inhalation toxicity study, groups of 5 male and 5 female young adult Fischer 344 rats were exposed to a vapor atmosphere of DPnB at concentrations of 0, 20, or 40 ppm (equivalent to 0, 160, or 320 mg/m3), by nose-only exposure.  Rats were exposed on weekdays 6 hr/day, 5 day/wk, for total of 9 exposures over a 2-week period.  
Polycarbonate tubes containing the subjects (nose cones) were attached to a 42-liter ADG nose-only cylindrical inhalation chamber (30 x 60 cm) with an airflow of 25 liters/min.  DPnB vapor was generated with the use of a J tube assembly, referenced (Miller et al. 1980. Am Ind Hyg Assc J. 41:844), but not described in the report.  Atmospheres were measured hourly by GC with a flame ionization detector.  Temperature and humidity were monitored at the end of each exposure period and the chamber was maintained at approximately 22°C and 50% relative humidity.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual chamber concentration was measured approximately once per hour using aVarian Aerograph 2400 gas chromatograph equipped with a stainless steel column packed with 10% OV-101 on 100/120 mesh Chromosorb W-HP at 190 celcius degree, with a flame ionization detector at 260 celcius degree and helium carrier gas. The analytical equipment was standardized by vaporizing measured volumes of the test substance in 50 liter Teflon bags filled with a measured volume of air. The concentration of DpnB in each chamber was then determined by interpolation from a standard curve. Standardization of the analytical system was evaluated with aat least one standard of known concentration prior to each exposure.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
9 exposures, 6 h exposure
Remarks:
Doses / Concentrations:
0, 20, 40 ppm (0, 0.16, 0.32 mg/l)
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: None
Positive control:
none
Observations and examinations performed and frequency:
Rats were observed after each exposure for mortality and clinical signs of toxicity.  The subjects were weighed prior to exposure on days 1, 3, 5, and 9 of the study.  Hematology, clinical chemistry, and urinalyses were conducted prior to sacrifice. 
Sacrifice and pathology:
All animals were subjected to gross necropsy and over 50 tissues were collected and processed into slides for histological examination.
Other examinations:
none
Statistics:
Descriptive statistics (mean and standard deviation) were reported for chamber concentrations, temperature and relative humidity, and leukocyte differential counts. Body weights, absolute and relative organ weights, clinical chemistry data, appropriate hematology data, and urine specific gravity were evaluated by Bartlett's test for equality of variances, parametric analysis of variance and Dunnett's test. Since multiple, interrelated parameters were statistically compared in the same group of animals, the frequency of false positive errors may be much greater than the nominal alpha levels. Therefore, in addition to the statistical analyses, the final toxicologic interpretation of the data considered factors such as dose-response relationships and biological plausibility.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
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:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Survival: All rats survived all nine exposures over the 14-day study period.  

Clinical signs: Some rats displayed porphyrin staining around the nares and were soiled with urine and feces from being in the nose cones after exposures in the first half of the study.  The authors attributed this to the stress of confinement in the polycarbonate tubes. 

Body weights did not increase in any group, probably due to the stress of confinement.  No differences were noted in body weights when DPnB-exposed rats were compared to air-only controls.  

No gross pathological lesions were noted at necropsy.

Organ weights: Liver weights in females were slightly increased in the mid and high exposure groups but there was no clear dose-response and there were no associated histopathological alterations in this organ or in clinical chemistry parameters indicating damage to this organ, and was considered incidental.

Hematology:  DPnB-exposed males showed a statistical increase in RBCs (20 and 40 ppm), hematocrits (20 and 40 ppm), and hemoglobin (40 ppm only) when compared to controls.  These findings were not considered treatment related because 1) increases were slight, 2) values were within laboratory historical control ranges, 3) no signs were evident indicating dehydration (e.g., diarrhea, abnormal electrolytes, diuresis), and 4) erythropoiesis was not evident when slides were examined of spleen, bone marrow, liver, or lymphoid tissue.

Clinical Chemistry:  No changes were noted that were considered treatment-related.  Males in the 40-ppm group showed a slight statistical increase in albumin that may have been related to the increased hemoconcentration discussed above.  Regarding spurious changes obviously not related to treatment, males in the 20-ppm group showed a slight decrease in potassium and females in this group a slight increase in alkaline phosphatase activity.

Urinalysis:  The only change noted was an increased number of males in the 40-ppm group with occult blood (3 of 5) as opposed to the control (1 of 5) and 20 ppm group (1 of 5). This finding was not considered treatment related but rather due to mild trauma of handling (pressure was applied to the abdomen to obtain urine samples).

Histopathology:  No differences were noted between the 40-ppm group and controls.  Consequently, the 20-ppm group was not examined.
Dose descriptor:
NOAEL
Effect level:
40 ppm
Sex:
male/female
Basis for effect level:
other: highest attainable concentration
Dose descriptor:
LOAEL
Effect level:
> 40 ppm
Sex:
male/female
Basis for effect level:
other: highest attainable concentration
Critical effects observed:
not specified
Conclusions:
DPnB did not cause toxicity by the inhalation (nose-only) route of exposure in Fischer 344 rats at atmospheric concentrations up to and including 40 ppm when exposed 6 hr/day on 9 separate days (over a 2-week period). The NOAEL is 40 ppm (highest attainable concentration) and the LOAEL was not established.
Executive summary:

In a 2-week inhalation toxicity study, groups of 5 male and 5 female young adult Fischer 344 rats were exposed to a
vapor atmosphere of DPnB at concentrations of 0, 20, or 40 ppm (equivalent to 0, 160, or 320 mg/m3), by nose-only
exposure.  Rats were exposed on weekdays 6 hr/day, 5 day/wk, for total of 9 exposures over a 2-week period.  Rats were
observed after each exposure for mortality and clinical signs of toxicity.  The subjects were weighed prior to exposure on days 1, 3, 5, and 9 of the study.  Hematology, clinical chemistry, and urinalyses were conducted prior to
sacrifice.  All animals were subjected to gross necropsy 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 cylindrical
inhalation chamber (30 x 60 cm) with an airflow of 25 liters/min.  DPnB vapor was generated with the use of a J
tube assembly, referenced (Miller et al. 1980. Am Ind Hyg Assc J. 41:844), but not described in this report. 
Atmospheres were measured hourly by GC with a flame ionization detector.  Temperature and humidity were
monitored at the end of each exposure period and the chamber was maintained at approximately 22°C and 50% relative
humidity.


Age at dosing:        At least 5 weeks of age.
Source:               Charles River Breeding Laboratory,Kingston, N.Y.
Acclimation period:   At least one week.
Weight range 
(start of study):     Males: 206 to 224 grams; 
                      Females: 139 to 152 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 in glass jars.
Access to water:      Available ad libitum (glass bottles).
Method of 
Identification:       Ear tags.
Housing:              Individually in stainless steel cages
                      with wire-mesh bottoms during
                      non-exposure periods. In poly- 
                      carbonate tubes during daily 6-hr
                      exposures.  Animals were acclimated
                      to the tubes 4 days prior to exposure
                      (1 hr/ on day -4, 3 hr on day -3, and
                      6 hrs -2 and -1).

Environmental Conditions (for nonexposure periods):

Temperature:          Approximately 22°C (recorded at the
                      end of each exposure period).   
Humidity:             50% (recorded at the end of each
                      exposure period).
Air changes:          not specified.
Photoperiod:          12 hr light/12 hr dark.

Environmental Conditions (for exposure periods):

Temperature:          Approximately 22°C (recorded at the
                      end of each exposure period).
Humidity:             50% (recorded at the end of each
                      exposure period).
Air changes:          >25 air changes per hour.
Photoperiod:          12 hr light/12 hr dark.

Survival: All rats survived all nine exposures over the 14-day study period.  Clinical signs: Some rats displayed
porphyrin staining around the nares and were soiled with urine and feces from being in the nose cones after exposures
in the first half of the study.  The authors attributed this to the stress of confinement in the polycarbonate tubes. 
Body weights did not increase in any group, probably due to the stress of confinement.  No differences were noted in
body weights when DPnB-exposed rats were compared to air-only controls.  No gross pathological lesions were noted
at necropsy.

Organ weights: Liver weights in females were slightly increased in the mid and high exposure groups but there was
no clear dose-response and there were no associated histopathological alterations in this organ or in clinical
chemistry parameters indicating damage to this organ. 

Hematology:  DPnB-exposed males showed a statistical increase in RBCs (20 and 40 ppm), hematocrits (20 and 40
ppm), and hemoglobin (40 ppm only) when compared to controls.  These findings were not considered treatment
related because 1) increases were slight, 2) values were within laboratory historical control ranges, 3) no signs
were evident indicating dehydration (e.g., diarrhea, abnormal electrolytes, diuresis), and 4) erythropoiesis was
not evident when slides were examined of spleen, bone marrow, liver, or lymphoid tissue.

Clinical Chemistry:  No changes were noted that were considered treatment-related.  Males in the 40-ppm group
showed a slight statistical increase in albumin that may have been related to the increased hemoconcentration
discussed above.  Regarding spurious changes obviously not related to treatment, males in the 20-ppm group showed a
slight decrease in potassium and females in this group a slight increase in alkaline phosphatase activity.

Urinalysis:  The only change noted was an increased number of males in the 40-ppm group with occult blood (3 of 5) as
opposed to the control (1 of 5) and 20 ppm group (1 of 5). This finding was not considered treatment related but rather
due to mild trauma of handling (pressure was applied to the abdomen to obtain urine samples).

Histopathology:  No differences were noted between the 40-ppm group and controls.  Consequently, the 20-ppm group
was not examined.


DPnB at 40 ppm was considered to be the highest practical concentration that could be obtained.  Nominal
concentrations (calculated by dividing the amount of DPnB consumed by the volume of air that flowed through the
chamber) agreed well with actual (measured) concentrations at 20 ppm but were considerably higher at 40 ppm, indicating
condensation and having reached the practical maximum concentration.

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 (in this limit test)
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.

DPnB did not cause toxicity by the inhalation (nose-only) route of exposure in Fischer 344 rats at atmospheric concentrations up to and including 40 ppm when exposed 6 hr/day on 9 separate days (over a 2-week period). The NOAEL is 40 ppm and the LOAEL was not established.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
200 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
acceptable

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
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
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
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.
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)
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
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.
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.
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
Critical effects observed:
not specified
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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
Study duration:
subacute
Species:
rat
Quality of whole database:
acceptable

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May 1987-February 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 411
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Principles of method if other than guideline:
n/a
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar (Bor: WISW (SPF Cpb))
Sex:
male/female
Details on test animals and environmental conditions:
See details below
Type of coverage:
other: non-occluded application, covered with collars
Vehicle:
propylene glycol
Details on exposure:
Dipropylene glycol n-butyl ether (DPnB) was applied daily (5 days/week) for 13 weeks to the skin of four groups of Wistar rats (10/sex/dose level) at various dilutions in propylene glycol (PG) equivalent to doses of 0 (PG-only; 1.5 ml/kg-day), 0.1, 0.3, or 1.0 ml DPnB/kg-day.  These doses equate to 0, 91, 273, or 910 mg DPnB/kg-day.  Treatment solutions were applied to the clipped dorsal trunk of each rat.  Dilutions of DPnB in PG resulted in applied volumes of 1.5 to 2.5 ml test solution per kg body weight.  Rats wore collars to prevent grooming and ingestion of test material.  Solutions were applied unoccluded since the low vapor pressure of DPnB and PG precluded evaporative loss.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of DPnB was determined in the solutions prepared on 11-5-87, 22-5-87, 5-6-87 and 3-7-87. The left-overs of the test solutions prepared on 22-5-87 were reanalyzed after storage for 2 weeks under the experimental solutions. The analyses were carried out at TNO, Institute CIVO-Analysis.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days/week
Remarks:
Doses / Concentrations:
0, 91, 273, or 910 mg/kg bw-day (0.1, 0.3, or 1 ml/kg bw-day)
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
Post-exposure period: none
Positive control:
none
Observations and examinations performed and frequency:
Rats were observed for clinical signs of toxicity and skin reactions on a daily basis (week days).  Body weights and food consumption were monitored weekly.  Ophthalmological examinations were conducted in control and high dose subjects prior to treatment and on day 85 of the study. Hematology, clinical chemistries, and urinalyses were conducted at the end of the treatment period.  
Sacrifice and pathology:
At sacrifice, all animals were subjected to complete necropsy.  An extensive list of tissues was preserved from all animals and histopathological evaluations of these tissues were conducted on control and high dose animals. 
Other examinations:
n/a
Statistics:
Data on body weights were evaluated by one-way analysis of co-variance followed by Dunnett's multiple comparison test. Data on food intake, food efficiency, red blood cell variables, total white blood cells, clinical chemistry values, volume and density of the urine, and organ weights were evaluated by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. Differential white blood cell counts were analyzed by the Mann-Whiteney U-test. The results of ophthalmoscopic examination and the histopathological changes were examined by Fisher's exact probability test.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
Skin at the site of application showed irritation in all treatment groups including PG-controls.  Grossly, irritation appeared as erythema, edema, scaliness, incrustations, and superficial scar tissue.  Skin lesions were characterized microscopically by focal necrosis of the epidermis, crust formation, mild inflammatory changes and acanthosis.  These changes were more severe in the high DPnB-treatment group. Untreated skin was unaffected.  The authors considered skin lesions to be a direct, local effect from the solvents and the clipping procedure. One high-dose male with a palpable mass was removed from the study and later died.  Necropsy and microscopic analysis of this subject revealed an overfilled urinary bladder due to obstruction of the urinary tract.  This death was not deemed treatment-related.  

No changes were observed in clinical appearance or behavior.  

Body weights in mid and high-dose males were lower than controls from week three until the end of the study.  

Food consumption was slightly increased in high-dose females and food conversion efficiency in mid and high-dose males was lower than controls (conversion efficiency differences were not generally statistically significant).  

Ophthalmological examination showed no effect from DPnB treatment.  White cell counts (neutrophils) were increased in mid and high-dose males with a similar but lesser trend in females.  SGOT (ALT) and SGPT (AST) were increased in high-dose males and triglycerides were increased in high-dose females.  Also, glucose was decreased in high-dose females. 

Urinalyses revealed no differences between control and DPnB-treated rats.  

Relative liver weights of both sexes were elevated in the high-dose group. 

No differences were noted between treated and control subjects from gross examination at necropsy.  Histopathology revealed the changes described above in the area of skin where treatment solutions were applied.  No other microscopic lesions were attributable to DPnB treatment.
Dose descriptor:
NOAEL
Effect level:
91 other: mg/kg
Sex:
male/female
Basis for effect level:
other: body weight changes and increased neutrophil counts at 273 mg/kg-day
Dose descriptor:
LOAEL
Effect level:
273 other: mg/kg
Sex:
male/female
Basis for effect level:
other: body weight changes and increased neutrophil counts
Critical effects observed:
not specified

none

Conclusions:
This study established a systemic toxicity NOAEL for DPnB of 0.1 ml/kg-day, or 91 mg/kg-day. A LOAEL, based on body weight changes and increased neutrophil counts, was 0.3 ml/kg-day or 273 mg/kg-day. No evidence was found for a hemolytic effect from DPnB treatment.
Executive summary:

Dipropylene glycol n-butyl ether (DPnB) was applied daily (5 days/week) for 13 weeks to the skin of four groups of Wistar
rats (10/sex/dose level) at various dilutions in propylene glycol (PG) equivalent to doses of 0 (PG-only; 1.5
ml/kg-day), 0.1, 0.3, or 1.0 ml DPnB/kg-day.  These doses equate to 0, 91, 273, or 910 mg DPnB/kg-day.  Treatment
solutions were applied to the clipped dorsal trunk of each rat.  Dilutions of DPnB in PG resulted in applied volumes of
1.5 to 2.5 ml test solution per kg body weight.  Rats wore collars to prevent grooming and ingestion of test material. 
Solutions were applied unoccluded since the low vapor pressure of DPnB and PG precluded evaporative loss.

Rats were observed for clinical signs of toxicity and skin reactions on a daily basis (week days).  Body weights and
food consumption were monitored weekly.  Ophthalmological examinations were conducted in control and high dose
subjects prior to treatment and on day 85 of the study. Hematology, clinical chemistries, and urinalyses were conducted at the end of the treatment period.  At sacrifice, all animals were subjected to complete necropsy.  An
extensive list of tissues was preserved from all animals and histopathological evaluations of these tissues were conducted on control and high dose animals.
 

.

Skin at the site of application showed irritation in all treatment groups including PG-controls.  Grossly, irritation appeared as erythema, edema, scaliness, incrustations, and superficial scar tissue.  Skin lesions were characterized
microscopically by focal necrosis of the epidermis, crust formation, mild inflammatory changes and acanthosis.  These
changes were more severe in the high DPnB-treatment group. Untreated skin was unaffected.  The authors considered skin
lesions to be a direct, local effect from the solvents and the clipping procedure.

One high-dose male with a palpable mass was removed from the study and later died.  Necropsy and microscopic analysis of
this subject revealed an overfilled urinary bladder due to obstruction of the urinary tract.  This death was not deemed
treatment-related.  No changes were observed in clinical appearance or behavior.  Body weights in mid and high-dose
males were lower than controls from week three until the end of the study.  Food consumption was slightly increased in
high-dose females and food conversion efficiency in mid and high-dose males was lower than controls (conversion
efficiency differences were not generally statistically significant).  Ophthalmological examination showed no effect
from DPnB treatment.  White cell counts (neutrophils) were increased in mid and high-dose males with a similar but
lesser trend in females.  SGOT (ALT) and SGPT (AST) were increased in high-dose males and triglycerides were
increased in high-dose females.  Also, glucose was decreased in high-dose females.  Urinalyses revealed no differences
between control and DPnB-treated rats.  Relative liver weights of both sexes were elevated in the high-dose group. 
No differences were noted between treated and control subjects from gross examination at necropsy.  Histopathology
revealed the changes described above in the area of skin where treatment solutions were applied.  No other microscopic lesions were attributable to DPnB treatment.

Skin at the site of application showed irritation in all treatment groups including PG-controls.  Grossly, irritation appeared as erythema, edema, scaliness, incrustations, and superficial scar tissue.  Skin lesions were characterized
microscopically by focal necrosis of the epidermis, crust formation, mild inflammatory changes and acanthosis.  These
changes were more severe in the high DPnB-treatment group. Untreated skin was unaffected.  The authors considered skin
lesions to be a direct, local effect from the solvents and the clipping procedure.

This study established a systemic toxicity NOAEL for DPnB of 0.1 ml/kg-day, or 91 mg/kg-day. A LOAEL, based on body weight changes and increased neutrophil counts, was 0.3 ml/kg-day or 273 mg/kg-day. No evidence was found for a hemolytic effect from DPnB treatment.

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.  The study report followed OECD Protocol 411: "Subchronic Dermal Toxicity:
90-day Study," the numbers and type of test animals used and their husbandry conditions were as prescribed in the
guidance.  Test material characterization was adequate. The amount of test material applied complied with guidance, the
length of the treatment period (90 days) was sufficient, and evaluation criteria and statistical methods were typical for
this type assay and adequately recorded.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
91 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
acceptable

Additional information

Oral:

Three oral toxicity studies are available for DPnB. The 90-day dietary study (using rats) has been selected as the key study for this endpoint as this is the longest exposure duration. The NOAEL is 450 mg/kg-day and the LOAEL is 1000 mg/kg-d, based on decreased body weights, increased liver weights (without accompanying histopathology) and slight alterations in clinical chemistry parameters. This is supported by the two 14 -day studies (oral gavage) with NOAELs of >400 and 750 mg/kg bw/day.

Dermal:

One 90 -d dermal toxicity study in rats is available. This study established a systemic toxicity NOAEL for DPnB of 91 mg/kg-day. A LOAEL, based on body weight changes and increased neutrophil counts, was 273 mg/kg-day. No evidence was found for a hemolytic effect from DPnB treatment. In this study there was a high incidence of skin irritation and in the mid and high dose groups. It is considered plausible that the stress caused by irritation could have led to depressions in bodyweight the local tissue damage could have contributed to increased neutrophil counts.

Inhalation:

Two inhalation toxicity studies are available for DPnB, one vapor inhalation study and one aerosol inhalation study.

In the vapor inhalation study, DPnB did not cause toxicity by the inhalation (nose-only) route of exposure in Fischer 344 rats at the highest attainable atmospheric concentrations of 40 ppm (~312 mg/m3) when exposed 6 hr/day on 9 separate days (over a 2-week period). The NOAEL is 40 ppm (~312 mg/m3) and the LOAEL was not established. In the aerosol inhalation study, the NOAEL for DPnB in this study was 200 mg/m3 and the LOAEL was 810 mg/m3 based on liver weight increase and irritation of the nasal mucosa.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
90-day oral study, well conducted and conforming to GLP

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
reliable study

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
study giving rise to highest concern

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
the only 90-day study available via the dermal route

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Repeated dose toxicity: inhalation - systemic effects (target organ) digestive: liver; respiratory: nose

Repeated dose toxicity: dermal - systemic effects (target organ) digestive: liver; other: skin

Justification for classification or non-classification

The no observed adverse effect levels for Dipropylene glycol n-butyl ether exceed the values triggering classification via all routes of exposure. Therefore no classification for prolonged exposure is required.