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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
repeated dose toxicity: oral
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, well-performed and documented study, performed in accordance with NTP guidelines, acceptable for assessment; however, chosen dose levels vastly exceeded the current limit doses recommended by OECD and EPA guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Principles of method if other than guideline:
Drinking water exposure of the male and female rats (50/sex/dose) to 0, 2500, 10000 and 40000 ppm dipropylene glycol for 105 weeks.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dipropylene glycol
- Substance type: organic
- Physical state: clear colorless liquid
- Analytical purity: ca. 99%
- Impurities (identity and concentrations): 0.475% water
- Isomers composition: ratio of 1,1'-oxybis(2-propanol) : 2-(2-hydroxypropoxy)-1-propanol : 2, 2'-oxybis(1-propanol) = 29 : 53 : 18
- Lot/batch No.: 10626DN
- Supplier: Aldrich Chemical Company, Inc. (Milwaukee, WI)
- Stability under test conditions: stable
- Storage condition of test material: at room temperature, protected from light, in amber glass containers.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6 weeks
- Weight at study initiation: 100-104 g (males), 93-94 g (females)
- Housing: 2-3 (males) or 5 per cage in solid-bottom polycarbonate (Lab Products, Inc., Maywood, NJ), with Sani-Chips bedding, changed twice weekly
- Diet: irradiated NTP-200 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA), ad libitum, changed weekly
- Water (e.g. ad libitum): tap water (Columbus municipal supply) via amber glass bottles with plastic Teflon-lined caps and stainless steel sipper tubes, ad libitum, changed twice weekly
- Acclimation period: 11 (males) or 12 (females)


ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3 F
- Humidity (%): 50% ± 15%
- Air changes (per hr): 10/hour
- 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:
Dose formulations were prepared every 7 to 12 weeks by mixing dipropylene glycol with tap water. Dipropylene glycol was added to tap water and mixed with a mechanical stirrer for approximately 5 minutes, then further diluted and stirred for an additional 5 minutes. Dose formulations were prepared approximately every 7 to 12 weeks. Dose formulations were stored in stainless steel drums at room temperature for max. 37 days.


VEHICLE
Water
- Concentration in vehicle: 0, 0.25, 1 and 4 mg/ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of dose formulations were conducted by the study laboratory using GC with flame ionization, RTX-5 fused silica, 15 m × 0.53 mm, 0.5-μm film (Restek, Bellefonte, PA), helium at 5 mL/minute, oven temperature program 50° C for 2 minutes, then 8° C/minute to 200° C. The dose formulations were analyzed approximately every nine weeks. Of the dose formulations analyzed, all 33 dose formulations were within 10% of the target concentrations. Animal room samples were also analyzed periodically; 11 of 12 samples for rats were within 10% of the target concentrations.
Duration of treatment / exposure:
105 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 2500, 10000 and 40000 ppm
Basis:
other: target in vehicle
Remarks:
Doses / Concentrations:
115, 470 and 3040 mg/kg bw/day (males), 140, 530 and 2330 mg/kg bw/day (females)
Basis:
actual ingested
No. of animals per sex per dose:
50/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the markedly decreased body weights in males and femals and the increased incidences of renal lesions and presence of atypical hepatocellular foci in males in 3-month study, 80000 ppm was considered too high for use in a 2-year study. Therefore the exposure concentrations for the 2-year drinking water study were 0, 2,500, 10,000, and 40,000 ppm. Although the incidences of renal lesions were increased in 40,000 ppm males in the 3-month study, the severities were minimal, and the lesions were not considered a potential threat to the health of the rats during a 2-year study. A wider exposure concentration range (fourfold steps) was used for the 2-year study because increased absolute and relative liver weights occurred at concentrations as low as 10,000 ppm in the 3-month study.
Note: the administered doses greatly surpassed the currently established limit doses according to OECD and EPA guidelines.
- Rationale for animal assignment (if not random): animals were distributed randomly into groups of approximately equal initial mean body weights

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical findings were recorded on day 36, monthly thereafter, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed initially, on days 8 and 36, monthly thereafter, and at the end of the studies.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water consumption was measured over a 7-day period at 4-week intervals, beginning the first week of the study.





Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsies were performed on all animals.
HISTOPATHOLOGY: Yes
- Complete histopathology was performed on all animals. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, heart and aorta, large intestine (cecum, colon, and rectum), small intestine (duodenum, jejunum, and ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.
Statistics:
Survival analyses: product-limit procedure of Kaplan and Meier
Neoplasm and nonneoplastic lesion prevalence: the Poly-k test
Organ, body weight, hematology and clinical chemistry data: parametric multiple comparison procedures of Dunnett (1955) and Williams (1971)
Spermatid and epididymal spermatozoal data: multiple comparison methods of Shirley (1977) and Dunn (1964)
Significance of the dose-related trends: Jonckheere's test (1954)
Treatment effects: multivariate analysis of variance (Morrison, 1976)

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Survival of 40,000 ppm males was significantly less than that of the control group. Survival of 40,000 ppm males declined steeply after week 58, and there were no survivors after week 98. Reduced survival was largely due to a high rate of moribund sacrifices that occurred between days 431 and 690; more than half of the sacrifices occurred between 18 and 24 months. Moribundity was probably caused by chronic nephropathy and subsequent renal insufficiency. Survival of males exposed to 2,500 or 10,000 ppm and all exposed groups of females was similar to that of the controls.
Moribund rats were lethargic and thin, and several breathed abnormally. There were no other chemical-related clinical findings.
Note: The significantly elevated mortality in the males in the 40,000 ppm group by the end of the study indicates that the dose level exceeded the maximally tolerated dose.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of male and female rats exposed to 40,000 ppm were less than those of the controls throughout the study. By week 94, group mean body weights of male and female rats exposed to 40,000 ppm were 28% and 15% less than those of the controls. Mean body weights of 2,500 and 10,000 ppm males and females were similar to those of the controls throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
During the first week of the study, water consumption by males and females exposed to 40,000 ppm was less than that by controls; the decrease was
attributed to taste aversion. However, water consumption by 40,000 ppm males increased after the first week of the study. From week 53 until the end of the study, water consumption by the 40,000 ppm males increased, resulting in an average consumption of 33.2 grams per day compared to 17.1 grams per day by the controls, suggesting renal insufficiency. Water consumption by males exposed to 2,500 or 10,000 ppm and all exposed groups of females was generally similar to that by the controls. Drinking water concentrations of 2,500, 10,000, or 40,000 ppm resulted in average daily doses of approximately 115, 470, or 3,040 mg/kg to males and 140, 530, or 2,330 mg/kg to females. Based on body weight, water consumption, and exposure concentration of dipropylene glycol, average daily doses for 2,500 and 10,000 ppm males and all exposed groups of females were proportional
throughout the study. However, the average daily doses for male rats exposed to 40,000 ppm were greater than proportional and were attributed to increased water consumption.
HISTOPATHOLOGY: NON-NEOPLASTIC
Adrenal Medulla: The incidences of benign pheochromocytoma of the adrenal medulla in 2,500 and 10,000 ppm male rats were increased (0 ppm, 4/47; 2,500 ppm, 7/49; 10,000 ppm, 12/50; 40,000 ppm, 1/47). The incidence in 10,000 ppm males was significantly greater than that in the controls and was at the upper end of the historical range in controls (all routes) given NTP-2000 diet [100/903 (11% ± 6%), range 3%-24%]. However, the incidence of benign or malignant pheochromocytoma (combined) in 10,000 ppm males was not significantly increased (9/47, 7/49, 13/50, 1/47), indicating that the significant increase in the incidence of benign pheochromocytoma in 10,000 ppm males was not related to dipropylene glycol exposure. The incidence of benign or malignant pheochromocytoma (combined) in 40,000 ppm males was less than that in the controls. The biological significance of this effect is not clear but may have been related to the decreased survival in 40,000 ppm males. However, since most of the deaths occurred late in the study, mortality was unlikely to have completely masked an exposure-related effect.

Kidney: Although chronic nephropathy occurred in most male rats, including the controls, the incidences and severities in 10,000 and 40,000 ppm males were increased. Nephropathy was considered to be the cause of the debilitation that resulted in early moribund sacrifice of many 40,000 ppm males. Nephropathy is a common spontaneous age-related lesion in F344/N rats, particularly males, and occurs in virtually all male rats in NTP 2-year studies. Exacerbation of nephropathy is frequently observed as a treatment-related effect and is manifested as an increase in severity. According to the review of Hard and co-authors (Hard et al., 2009), rodent CPN has no strict counterpart in humans, therefore these findings were considered to be irrelevant for the human risk assessment. The incidences of transitional epithelial hyperplasia in 10,000 and 40,000 ppm males were significantly increased. Transitional epithelial hyperplasia was generally mild in severity and consisted of focal papillary or nodular proliferation of the transitional epithelium lining the renal pelvis and was considered a component of chronic nephropathy.
The incidences of parathyroid gland hyperplasia (0 ppm, 0/45; 2,500 ppm, 4/48; 10,000 ppm, 1/49; 40,000 ppm, 5/50) and heart mineralization (0/50, 0/50, 0/50, 7/49) were significantly increased in 40,000 ppm males. These lesions are considered to be secondary to chronic nephropathy. The microscopic lesions of nephropathy were generally similar to the spontaneous lesions (Plates 4, 5, and 6). The lesions were characterized by interstitial fibrosis and inflammation, renal tubule epithelial necrosis, and dilated renal tubules containing protein casts. Many cortical tubules contained dense eosinophilic material, and some were surrounded by neutrophils. Many glomeruli were enlarged with thickened basement membranes and capsules. Brightly eosinophilic hyaline material was also increased in the cortical and medullary interstitium and the glomerular mesangium.

Liver: The incidences of minimal to mild focal granulomatous inflammation of the liver were significantly increased in 10,000 and 40,000 ppm males. Although the mean severities of granulomatous inflammation were not different from that in the controls, more animals in these groups had a severity grade of mild. The incidence of granulomatous inflammation in males exposed to 40,000 ppm was less than in males exposed to 10,000 ppm, which may have been due to early deaths. The incidence of granulomatous inflammation in female rats exposed to 10,000 ppm was slightly increased; however, this increase was not significant and the severity was similar to that in the controls. Focal granulomatous inflammation was morphologically consistent with the spontaneous microgranulomatous lesions that are commonly observed in aged rats and considered to result from bacterial showering from the intestinal tract. This lesion occurred as small, randomly distributed foci predominantly composed of a mixture of small macrophages and lymphocytes with varying numbers of neutrophils (Plate 7). Larger foci tended to contain necrotic hepatocytes, minimal fibrosis, and slightly increased numbers of neutrophils.
There were exposure concentration-related increases in the incidences and severities of focal histiocytic inflammation in all exposed groups of male rats; the increases in the 10,000 and 40,000 ppm groups were significant. Focal histiocytic inflammation was also considered to be a spontaneous change, the morphology of which was clearly different from that of focal granulomatous inflammation. However, in control rats, foci of histiocytic inflammation were not as readily discernable as foci of granulomatous inflammation. The increased prominence and incidence of this lesion was considered an exacerbation of a background change in exposed animals. Histiocytic inflammation consisted of individual or multifocal small clusters of large, irregularly oval toround histiocytic cells that were primarily portal, periportal, and centrilobular in distribution, but occasionally were randomly distributed throughout the parenchyma (Plate 8). In the controls, the histiocytes occurred primarily as scattered infiltrates of individual cells and rarely as clusters. The histiocytes had abundant foamy to lightly basophilic, homogenous to finely granular cytoplasm that frequently contained one to numerous,
non-birefringent, irregularly elongate clear clefts (Plate 9). The numbers of cells with cytoplasmic clefts appeared to be prominently increased in the male rats exposed to 10,000 or 40,000 ppm. Occasionally, histocytes in the controls contained rare cleft-like structures that were not as prominent as those in treated animals. The contents of the clefts are unknown but, in the rats exposed to dipropylene glycol, some may have contained the tested material or its metabolized by-products. The cell nuclei were irregularly oval with lightly basophilic homogenous chromatin and indistinct nucleoli. Rare to frequent large syncytial cells with two or more nuclei were present; the nuclei were clustered or arranged individually around the periphery of the cell. The cytoplasm of some syncytial cells contained dense, homogenous, eosinophilic material that was usually centrally located.
The incidences of bile duct hyperplasia in 40,000 ppm males and females, basophilic foci in 2,500 and 40,000 ppm males, clear cell foci in 10,000 ppm females, and mixed cell foci in 2,500 and 10,000 ppm females were significantly greater than those in the controls. The incidences of clear cell foci and centrilobular necrosis in males exposed to 40,000 ppm were significantly less than those in the controls. The increased incidences of bile duct hyperplasia were considered related to dipropylene glycol exposure. Because the incidences of hepatocellular foci were variable and not exposure concentration related, they were unlikely related to dipropylene glycol exposure.

Nose: The incidences of minimal to moderate olfactory epithelial atrophy in 40,000 ppm male rats and of minimal to moderate olfactory degeneration in 40,000 ppm male and female rats were significantly greater than hose in the controls. The incidence of mild to marked thrombosis in males exposed to 40,000 ppm was significantly increased. Olfactory epithelial atrophy was a segmental change that involved the dorsal meatus in Level II and occasionally Level III. The lesion was characterized by segmental disorganization and decreases in the height and number of layers of epithelial cells with occasional individual cell necrosis. Olfactory epithelial degeneration was morphologically similar to that observed in the 3-month study. Degeneration affected the olfactory epithelium primarily in the dorsal meatus in Level II and segments of the olfactory epithelium in the ethmoid region (Level III) of the nasal cavity. In affected segments of the epithelium, large clear cytoplasmic vacuoles distorted many of the sustentacular cells. The biological significance of these nasal lesions is not certain but could be related to metabolism of dipropylene glycol in the olfactory epithelium. The olfactory epithelium of rats has a moderately welldeveloped enzyme system that includes enzymes of the cytochrome P450 family that are capable of metabolizing
xenobiotic chemicals.

Salivary Gland: The incidence of minimal to mild suppurative inflammation of the salivary gland was significantly increased in 40,000 ppm males [0 ppm, 0/49; 2,500 ppm, 1/49 (1.0); 10,000 ppm, 0/50; 40,000 ppm, 22/50 (1.8)]. The biological significance of this lesion is uncertain.

Mammary Gland: There was a significant decrease in the incidence of mammary gland fibroadenoma (36/50, 35/50, 30/50, 22/50) in 40,000 ppm females. This decrease was likely associated with the decreased body weight in this exposure group, because the incidence of mammary gland fibroadenoma in female rats is significantly correlated with changes in body weight (Haseman, 1983; Rao et al., 1987), and because the unusually high concurrent control value is outside the historical range for controls (all routes) given NTP-2000 diet [401/909 (42.1% ± 10.0%), range 28%-56%].

Forestomach: The incidences of forestomach ulceration (3/50, 5/50, 8/50, 10/49) and associated hyperplasia (0/50, 1/50, 3/50, 5/49) were increased in treated male rats. The biological significance of these changes in relation to exposure is uncertain.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
470 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: Effects in liver (increased incidence of bile duct hyperplasia) and nose (increased incidence of olfactory epithelial artrophy and degeneration)
Dose descriptor:
NOAEL
Effect level:
530 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: Effects in liver (increased incidence of bile duct hyperplasia) and nose (increased incidence of olfactory epithelial degeneration)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion