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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09/1990-04/1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study according to OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Dowanol-PnB (1-butoxy-2-hydroxypropane or
propylene glycol normal-butyl ether). CAS # 29387-86-8
(also 5131-66-8).
Batch No.: EB 891121.
Purity: 99.4% (96.17% 1-butoxy-2-propanol; 3.23%
2-butoxy-1-propanol).
Supplied as: Not reported.
Appearance: Clear liquid.
Administered as: Dilution in water.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Kingston, N.Y.
- Age at study initiation: at least 5 weeks
- Weight at study initiation: 105 to 110 g (males) and 87 to 89 g (females)
- Fasting period before study:
- Housing: Individually in stainless steel cages with wire-mesh bottoms.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum (with test material)
- Acclimation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Approximately 72°F
- Humidity (%): no data
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

A premixed solution (test material - water concentrate) to a determined amount of tap water. Test solutions and premixes were prepared weekly to maintain the the targeted concentrations as determined in the one week probe study.

Initial concentrations of test material were in the water were calculated from pretest body weights and water consumption data targeted on the desired dose levels on a mg/kg bw/day basis. Thereafter, the most recent body weight and water consumption data was used to adjust the concentration of the test material in the water to maintain those targeted dose levels. Water concentrations were adjusted weekly to maintain the targeted exposure levels.

Concentrations of PnB in drinking water were targeted to correspond to nominal doses of 0, 100, 350, or 1000 mg/kg-day.  These concentrations were based on anticipated drinking water consumption volumes by the test animals.  When the measured concentration of PnB in the treatment water was multiplied by the amount of water actually consumed, the actual dose of PnB was higher than the target doses (by a factor of 10-15%).  However, wastage may have occurred that would reduce the dose and offset the 10-15% increased dose factor.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of premix, test material/water mixtures, and municipial tap water taken on Study Day 1, Study Day 29, Study Day 57, and Study Day 85 were analyzed to confirm targeted concentrations of PnB in the water.

The samples were diluted with methanol (2/5) and analyzed by high pressure liquid chromatography using refractive index detection (HPLC).
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: the high dose of 1000 mg/kg bw/day was considered adequate for a limit test as definedby OECD. The lower dose levels were selected to establish a dose-response for potential effects and to determine the no-observed-effect-level (NOEL) and the no-observed-adverse-effect-level (NOAEL).
- Rationale for animal assignment: animal were weighed and randomly allocated to study groups using a computerized, weight-stratification and random number-based procedure. 
- Post-exposure recovery period: 4 weeks
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: including evaluation of the skin, fur, and mucous membranes, respiration, nervous system, and behaviour patterns. bservations on weekends and holidays were limited to a check of all cages for dead animalss and the availability of feed and water.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed for clinical signs of toxicity on a daily basis (week days). Particular attention was directed to observations for possible tremeors, convulsions, lethargy and other signs related to central nervous system function, salivation and diarrhea.

BODY WEIGHT: Yes
- body weights were monitored weekly.

FOOD CONSUMPTION:
- food consumption was monitored weekly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- body weights, water, and food consumption were monitored weekly.

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmological examinations were conducted prior to treatment and at sacrifice.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: hematology and electrolytes were evaluated at sacrifice.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes (overnight before sacrifice)
- Parameters examined: packed cell volume, hemoglobin concentration, red and white blood cell counts, platelet counts, and WBC differential counts.
Complete blood smear examinations were conducted which included differential leukocyte counts (the number of leukocytes counted to be specified if other than 100 cells were counted) and an assessment of erythrocyte, leukocyte, and platelet morphology.

CLINICAL CHEMISTRY: Yes
Clinical chemistries were evaluated at sacrifice.
- Time schedule for collection of blood:
- Animals fasted: Yes (over night before sacrifice)
- Parameters examined: alanine aminotransferase, alkaline phosphatase, aspartate aminitransferase, blood urea nitrogen, cholesterol, triglycerides, creatinine, creatine phosphokinase, total bilirubin, glucose, total protein, calcium, phosphorus, chloride, sodium, potassium, and albumin using a Spectrum Analyzer. The globulin concentrations were celaculated from the total protein and albumin values.

URINALYSIS: Yes
- Time schedule for collection of urine: urinalyses were conducted one week prior to sacrifice
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters examined: specific gravity, pH, bilirubin, glucose, protein, ketones, blood, and urobilinogen. In addition, the microsediment from a pooled sample from each group was examined microscopically.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional observational battery evaluations were conducted prior to treatment and at monthly intervals during treatment.

Sacrifice and pathology:
At sacrifice, all surviving animals were subjected to complete necropsy. A complete histologic evaluation was made on all control and high dose animals. Selected organs evaluated histologically in lower dose subjects included liver, kidneys, adrenal glands, lungs, testes, and, potentially, other target organs identified in high dose animals.
Other examinations:
none
Statistics:
Hematology (excluding differential counts), electrolyte, clinical chemistry data, body weights, and absolute (grams) and relative (g to 100g terminal body weight) organ weights were evaluated by Bartlett's test for equality of varaiances. Based on the outcome of Bartlett's test, exploratory data analysis was performed by a parametric or non-parametric analysis of variance (ANOVA), followed, if appropriate, by Dunnett's test or Wilcoxon rank-sum test with Bonferroni's correction for multiple comparisons. Statistical outliers were identified by a sequential test described by Grubbs. Feed and water consumption data, which were used in the computation of desired test material concentrations and shown in this final report, were not analyzed for differences of statistical significance.
Differences found to be statistically significant were not necessarily accepted as toxicologically significant, i.e., final interpretation of the numerical data did consider the statistical outcomes together with other factors, such as dose-response relationships, biologic plausibility, and pathological observations.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
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):
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:
CLINICAL SIGNS AND MORTALITY
All rats survived treatment with the test compound.

BODY WEIGHT AND WEIGHT GAIN
Slight decrease (less than 5% but statistically significant) in high dose males on days 21 through day 42. Correlated to reduced water and food intake.

FOOD CONSUMPTION
Slight decrease in high dose males and females (not statistically evaluated).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Slight decrease in high dose males and females (not statistically evaluated).

OPHTHALMOSCOPIC EXAMINATION
No lesions noted.

HAEMATOLOGY
Statistically decreased red blood cell count and hemoglobin in high-dose (1000 mg/kg-day) males at 13-week sacrifice. Statistically decreased platelet count in high dose females after 13 weeks. Statistically decreased platelet count in high dose recovery males. No corresponding hypertrophy or lesions in bone marrow or spleen for any group.

CLINICAL CHEMISTRY
In high dose males (1000 mg/kg-day), several parameters were statistically different from controls: Sodium (decrease), potassium (increase), chloride (decrease), creatine phosphokinase (increase), urea (increase), and cholesterol (increase). In the mid-dose group (350 mg/kg-day), slight statistically significant changes in sodium (decrease) and potassium (increase) were noted. High dose females showed slightly increased urea and creatine phosphokinase.

URINALYSIS
No abnormalities noted.

NEUROBEHAVIOUR
No behavioral effects noted.

ORGAN WEIGHTS
Absolute and relative liver weights were increased in high dose males with no accompanying histopathology. In females at the high dose level, absolute and relative kidney weights were increased with no accompanying histopathology. High-dose males showed increased absolute and relative liver weights. Females showed increased absolute and relative kidney weights. No corresponding histopathology was found in these organs.

GROSS PATHOLOGY

HISTOPATHOLOGY: NON-NEOPLASTIC
No lesions noted.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No lesions noted.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: PnB at doses of 1000 mg/kg-d for 13 weeks caused increased absolute and relative liver weights in males and increased absolute and relative kidney weights in females
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: increased absolute and relative liver weights in males and increased absolute and relative kidney weights in females.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
PnB at doses of 1000 mg/kg-d for 13 weeks caused increased absolute and relative liver weights in males and increased
absolute and relative kidney weights in females. The authors of the study concluded that the slight decreases in red blood cell count and hemoglobin in high-dose males may be related to decreased water and food consumption. This may also explain the slight decrease in male body weights
mid-way through exposures. Clinical chemistry changes, which may be similarly related to food and water consumption, were considered by the authors to be slight and not toxicologically significant. No effects from PnB were found at the lower dose levels of 100 or 350 mg/kg-day. The NOAEL for PnB is 350 mg/kg-day and the LOAEL is 1000 mg/kg-day.
Executive summary:

Four groups of Fischer 344 rats (10/sex/dose level) received propylene glycol n-butyl ether (PnB) in their drinking water at concentrations equivalent to target doses of 0, 100, 350,
or 1000 mg/kg-day for 13 weeks.  Two additional groups of 10/sex/dose receiving 0 or 1000 mg/kg-d for 13 weeks, were administered untreated water for four weeks following the 13-week exposure period in order to evaluate recovery.  Rats were observed for clinical signs of toxicity on a daily
basis (week days).  Body weights, water, and food consumption were monitored weekly.  Functional observational
battery evaluations were conducted prior to treatment and at monthly intervals during treatment.  Ophthalmological
examinations were conducted prior to treatment and at sacrifice.  Hematology, electrolytes, and clinical
chemistries were evaluated at sacrifice and urinalyses were conducted one week prior to sacrifice.  At sacrifice, all
control and high dose animals were subjected to complete necropsy and histopathological evaluations.  Selected organs evaluated histologically in lower dose subjects included
liver, kidneys, adrenal glands, lungs, testes, and, potentially, other target organs identified in high dose animals. 

Concentrations of PnB in drinking water were targeted to correspond to nominal doses of 0, 100, 350, or 1000
mg/kg-day.  These concentrations were based on anticipated drinking water consumption volumes by the test animals.  When the measured concentration of PnB in the treatment
water was multiplied by the amount of water actually consumed, the actual dose of PnB was higher than the target
doses (by a factor of 10-15%).  However, wastage may have occurred that would reduce the dose and offset the 10-15% increased dose factor.

Absolute and relative liver weights were increased in high dose males with no accompanying histopathology. 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 PnB is 350 mg/kg-day and the LOAEL is 1000 mg/kg-day (for organ weight changes).