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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06/1987-04/1988
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:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
GLP compliance:
yes (incl. certificate)

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.: "EH; dated 22-4-'87 (use within one year)".
Purity: "more than 98%".
Specific Gravity: 0.88 kg/l.
Solubility: 6% in water; soluble in propylene glycol.
Storage: Ambient temperature in the dark.
Appearance: Clear liquid.
Administered as: Dilution in propylene glycol.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann - Institute for the Breeding of Laboratory Animals GmbH & Co. KG, Borchen West-Germany
- Age at study initiation: ca. 10 weeks
- Weight at study initiation: Males: 249 ± 2.9 grams; Females: 173 ± 1.5 grams.
- Fasting period before study: none
- Housing: individually in suspended stainless steel cages fitted with wire-screen floor, sides and front
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 40-85%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
open
Vehicle:
propylene glycol
Details on exposure:
TEST SITE
- Area of exposure: dorsal trunk of each rat
- coverage: area for dermal application was ca. 25-40 cm2)
- Type of wrap if used: no wrap (rats wore collars to prevent grooming and ingestion of test material)
- Time intervals for shavings or clipplings: weekly (every Friday)

REMOVAL OF TEST SUBSTANCE
- Contact time was 24 hr/day (skin was cleaned shortly before daily treatment).

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 ml
- Concentration (if solution): 6.3%, 16.7% and 40.0% (v/v) PnB in propylene glycol
- Constant volume or concentration used: yes (constant volume)

VEHICLE
- Justification for use and choice of vehicle (if other than water): good solubilty of PnB in propylene glycol

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (collars)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The content of PnB was determined in the solutions prepared on 1-6-87, 12-6-87, 10-7-87 and 24-7-87. The left overs of the test solutions prepared on 10-7-87 were reanalyzed after storage for 2 weeks under the epxerimental conditions, in order to determine the stability of PnB in the test solutions. The analyses were carried out at TNO, Institute CIVO-Analysis.

Samples were diluted with acetone and a known amount of the solution was injected in a gas chromatographic column. Detection was performed by means of a flame ionization detector. Quantitation was performed by means of a calibration curve of Propylene Glycol n-Butyl Ether.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
0.1 other: ml/kg bw
Remarks:
Basis: nominal per unit body weight
Dose / conc.:
0.3 other: ml/kg bw
Remarks:
Basis: nominal per unit body weight
Dose / conc.:
1 other: ml/kg bw
Remarks:
Basis: nominal per unit body weight
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: since PnB is considered a substance of low toxicity, a top dose level of 1 ml PnB/kg body weight was chosen.
- Rationale for animal assignment (if not random): Computerized, random number-based procedure.
- Post-exposure recovery period in satellite groups: no
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed daily and all signs of ill health and reaction to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed for clinical signs of toxicity on a daily basis (week days).

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Rats were observed for skin reactions on a daily basis (week days).

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights of all rats were recorded initially, and once every week thereafter. In addition, females were weighed on day 92 in order to calculate the correct organ to body weight ratios at the day of autopsy.

FOOD CONSUMPTION:
- Food intake was measured per animal weekly, and the efficiency of food utilization was calculated and expressed as gram weight gain per gram food consumed.

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at the beginning (day 0) and at the end of the (day 87)
- Dose groups that were examined: all rats of the control group and the high-dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 85 (males) and day 86 (females)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all surviving animals in the study
- Parameters examined: haemoglobin, packed cell volume, red blood cells, white blood cells, differential white blood cell count, thrombocytes, prothrombin time, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 88 (for glucose determination) and at autopsy (day 91 for males, day 92 for females)
- Animals fasted: Yes for glucose determination (24 h depriviation of water and 16 h depriviation of food)
- How many animals: all surviving animals in the study
- Parameters examined: alkaline phosphatase activity (ALP), glutamic-oxalacetic-transaminase (GOT)/aspartate amino transferase (ASAT) activity, glutamic-pyruvic transaminase (GPT)/alanine aminotransferase (ALAT) activity, gamma glutamyl transpeptidase activity, total protein, albumin, albumin/globulin ratio, urea, creatinine, bilirubin total, cholesterol, triglycerides, sodium (Na), potassium (K), calcium (Ca), chloride (Cl), inorganic phosphate

URINALYSIS: Yes
- Time schedule for collection of urine: day 88
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes (deprived of water for 24 hours and of food for 16 hours, urine collected during last 16 hours of depriviation period)
- Parameters examined: in individual samples (volume, density) and in pooled samples - one sample per sex per group (appearance, pH, protein, glucose, occult blood, ketones, bilirubin, uribilinogen, erythrocytes, leucocytes, epithelial cells, amorph material, crystals, casts, bacteria, sperm cells and worm eggs)

NEUROBEHAVIOURAL EXAMINATION: No
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:
none
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 variances (ANOVA) followed by Dunnett's multiple comparison test. Differential white blood cell counts were analysed by the Mann-Whitney U-test. The results of ophthalmoscopic and microscopic examinations were analysed by Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
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:
CLINICAL SIGNS AND MORTALITY
no deaths and no changes in clinical observations

BODY WEIGHT AND WEIGHT GAIN
no changes observed

FOOD CONSUMPTION
no changes observed

WATER CONSUMPTION
no data

OPHTHALMOSCOPIC EXAMINATION
no changes observed

HAEMATOLOGY
no changes observed

CLINICAL CHEMISTRY
no changes observed

URINALYSIS
no changes observed

NEUROBEHAVIOUR
no data

ORGAN WEIGHTS
In females at the high dose level, relative but not absolute heart weights were slightly but statistically significant increased. Because no clinical chemistry or histopathology indicated damage to the heart, the authors considered increased relative weights to be a spurious finding without toxicological significance.

GROSS PATHOLOGY
no changes observed

HISTOPATHOLOGY: NON-NEOPLASTIC
no changes observed

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
no changes observed

OTHER FINDINGS
Skin at the site of application showed irritation in all treatment groups including PG-controls. Skin lesions were characterized by focal necrosis of the epidermis, crust formation, mild inflammatory changes and acanthosis. While the severity of these lesions was higher in PnB-treatment groups than in the PG-control, the differences were not statistically significant and did not show a dose-response effect. Untreated skin was unaffected (one mid-dose male and one high-dose male showed “very slight” acanthosis). The authors considered skin lesions to be a direct, local effect from the solvents and the clipping procedure.

Effect levels

Dose descriptor:
NOAEL
Effect level:
880 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: no biologically significant observable adverse effects in rats.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The systemic toxicity NOAEL for PnB is 1.0 ml/kg-day, or 880 mg/kg-day, and the LOAEL was not established in this study.
Executive summary:

Propylene glycol n-butyl ether (PnB) 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 PnB/kg-day. These doses equate to 0, 88, 264, or 880 mg PnB/kg-day. Treatment
solutions were applied to the clipped dorsal trunk of each rat. Dilutions of PnB 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 PnB and PG precluded evaporative loss. Contact time was 24 hr/day (skin was cleaned shortly before daily treatment). 

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 87 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.  Skin lesions were
characterized by focal necrosis of the epidermis, crust formation, mild inflammatory changes and acanthosis.  While
the severity of these lesions was higher in PnB-treatment groups than in the PG-control, the differences were not
statistically significant and did not show a dose-response effect.  Untreated skin was unaffected (one mid-dose male
and one high-dose male showed "very slight" acanthosis).  The authors considered skin lesions to be a direct, local
effect from the solvents and the clipping procedure.

No changes were observed in clinical observations, food consumption, body weights, ophthalmology, hematology,
clinical chemistries, urinalyses, or gross lesions/histopathology (other than skin).  In females at the
high dose level, relative but not absolute heart weights were slightly but statistically increased.  Because no clinical chemistry or histopathology indicated damage to the heart, the authors considered increased relative weights to be a spurious finding without toxicological significance. 
This study established a systemic toxicity NOAEL for PnB of 1.0 ml/kg-day (880 mg/kg-day).  A LOAEL for systemic
toxicity was not established.