Oxydipropanol

Endpoint summary

Description of key information

NTP drinking water studies with rats and mice of different exposure durations (14-day, 90-day and 2-year) were available for assessment. The main effects were found in liver and kidneys. Increased incidence and severity of chronic progressive nephropathy (CPN) and subsequent renal insufficiency was observed in male rats exposed to 10,000 and 40,000 ppm dipropylene glycol in drinking water in the 2-year study. However, as rodent CPN is believed not to have a strict counterpart in humans, these findings were considered to be irrelevant for human risk assessment. Based on the results of 2-year carcinogenicity study with rats, the NOAEL for repeated dose toxicity was established to correspond to 470 and 530 mg/kg bw/day for male and female rats, respectively (actual ingested dose), based on the bile duct hyperplasia and olfactory epithelium degeneration and/or atrophy. These values shall be used for the risk assessment and DNEL derivation.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: oral

Remarks:

combined repeated dose and carcinogenicity

Type of information:

experimental study

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.

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

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system 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
:

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

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

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)

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.
The observed incidences were also comparable with the historical control data for chronic non-neoplastic lesions in F-344 rats in the 2-year NTP studies (Experimental Pathology Laboratories, Inc., 2008 ), and therefore regarded as irrelevant for human risk assessment.
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.

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)

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

470 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No studies using inhalation or dermal routes of exposure to dipropylene glycol were available for assessment.

Regarding the oral route of exposure, an NTP drinking water study in rats and mice, including 14-day, 90-day, and 2-year exposure duration, was available for assessment (National Toxicology Program, 2004). Based on the longest exposure duration and the lowest observed effect level, the 2 -year study with rats was chosen as a key study.

In the 90-day study with rats, animals were exposed to 0, 5000, 10000, 20000, 40000 or 80000 ppm in drinking water, resulting in actual ingested doses of 425, 890, 1840, 3890 and 12800 mg/kg bw/day (males) and 460, 920, 1690, 3340 and 8950 mg/kg bw/day (females). It should be noted that these doses vastly exceeded the limit dose values currently established by OECD and EPA guidelines. Two test groups were used, a core group and a clinical pathology group, each including 10 animals/sex/dose. In addition to gross necropsy, histopathological and clinical chemistry examinations conducted, at the end of the studies, sperm motility and estrous cycle evaluations were conducted on the animals in the 0, 5,000, 20,000, and 80,000 ppm groups.

The main effects were observed in liver and kidneys. Exposure to 10,000 ppm or greater caused significant increases in absolute and relative liver weights of males and females compared to those of the controls. Exposure to 20,000 ppm or greater in males and 40,000 ppm or greater in females caused significant increases in relative kidney weights. In male rats exposed to 80,000 ppm, the incidence of foci of hepatocellular alteration, classified histologically as atypical hepatocellular foci, was significantly increased. The left testis, cauda epididymis, and epididymis weights; motility of epididymal spermatozoa; epididymal sperm counts per cauda; and spermatid heads per gram testis of 80,000 ppm males were significantly decreased. No significant differences were noted in estrous cycle parameters between exposed and control females. The toxicological relevance of the male reproductive tissue findings is however questionable, as it is obvious that the doses ingested by the study animals were excessively high, surpassing the currently by OECD and EPA established limit. The established NOAELs were 425 mg/kg bw/day for male and 460 mg/kg bw/day for female rats.

The testicular and sperm effects noted at the 80,000 ppm group require analysis and discussion. The onset of puberty and sexual maturity is affected by age of the animal, body mass, growth rate and development. Nutritional deficiencies can delay the onset of puberty and sexual maturation as well as affect the growth and development of the gonads and accessory sex organs (Hamm et al. 1983; Chapin et al. 1993).Although the feed consumption values were not provided in the NTP study, feed consumption values typically mirror water consumption values in rodent species (Duffy et al. 1989). Prior to puberty, although there is continued differentiation, growth of the testes and accessory sex glands mirror somatic growth (Marty et al., 2006). With the onset of puberty, the growth of the testes and accessory sex organs are disproportionately increased when compared to somatic growth, primarily due to the growth stimulatory effects of gonadotropins and steroid sex hormones. Nutritional deficiencies (e.g. reduced feed and/or water intake) prior to puberty affect growth of the testes and sex organs as these conditions affect somatic growth. Decreases in feed and/or water consumption before puberty can also delay the onset of sexual maturation endpoints and this is why both age and body weight are collected on the day of attainment of sexual maturation endpoints (e.g. balanopreputial separation, vaginal opening and estrus cycling) in guideline regulatory studies.

However, after sexual maturity is attained, these same nutritional deficiencies (e.g. feed and/or water restriction) do not affect the growth of the testes and accessory sex organs as it does somatic growth (Holson et al. 2006). This phenomena is sometimes referred to as “testicular sparing” and describes a lack of effect of feed and/or water restriction on testes and accessory gland weights. This finding is commonly observed in repeated-exposure studies when the animals are sexually mature at the onset of the exposure period and following a period of feed and/or water restriction, the testes weights are comparable to the control group values and the relative testes weights (relative to body weight) are increased, due to the decreased overall growth of the animal. 

The 90-day rat NTP study used sexually immature F344 male rats that were 6 weeks of age at the start of the exposure. F344 male rats become sexually mature between ten and fifteen weeks of age (Blazak et al. 1985), providing normal feed consumption, body weight gains and growth patterns. However, in the NTP 90-day study, the high-dose rats consumed 40% less water during the first week of exposure when compared to controls. These decrements in water consumption (and presumably feed consumption although the data was not provided) continued throughout the exposure period to varying degrees resulting in terminal body weights in the male rats that were 53% that of the control group. Decrements in feed and water consumption (resulting in a severe decrease in rate of body weight gain) during the period of sexual maturation and growth can delay (or even prevent) the onset of puberty in male rats. This problem of feed restriction affecting sexual development is discussed in Chapin et al. (1993) and Hamm et al. (1983). Chapin et al. (1993) noted the differing response in Sprague Dawley and F344 rat strains as well as the effect of feed restriction prior to sexual maturity. The comparison suggests that 1) F344 rats (an inbred strain) are more susceptible to reproductive organ weight changes than Sprague Dawley rats (an outbred strain) and 2) that feed restriction prior to sexual maturity affected sex organ weight changes and sexual function more severely than the same extent of feed restriction that commenced after sexual maturity was attained.

With the extent of the water intake reduction and probably feed restriction noted in the 80,000 ppm group in combination with the resultant effect on male rat body weights (53% that of the control group values), it is concluded that the effects noted were the result of a primary cause from nutritional deficiencies with subsequent effects on immature testes and sex organs during the 90-day exposure period. This delay in attaining sexual maturity is therefore secondary to a nutritional and growth deficiency and does not represent an endocrine-mediated insult.

An additional consideration in the use of sexually immature F344 rats in this study is the lack of a full complement of adult metabolic enzymes required to metabolize and eliminate the administered test chemical during the first few weeks of the study. If the animals are growth-retarded from lack of adequate nutrition (with the decrements in water/feed consumption and dramatic decreases in body weight gain as evidence), then the maturation processes necessary to provide a full complement of adult metabolic enzymes (in the liver as well as other tissues) could very well be delayed. The concept of “maximally tolerated dose“ (MTD) should be dependent upon developmental life stage as the full complement of adult metabolic enzymes are not present until the animal becomes fully sexually mature. If sexual maturation is delayed from nutritional deficiencies, then the development of a full complement of adult metabolic enzymes would be expected to be delayed as a secondary consequence of the reduced nutrient intake. It is concludedfrom the dramatic decrease in body weight gain parameters observed in the 80,000 ppm dose level male rats that the MTD was clearly exceeded in these sexually immature animals.

In the 90-day mice study, the exposure concentrations were the same as in the rat study, resulting in actual ingested concentrations of 715, 1350, 2620, 4790 and 11000 mg/kg bw/day (males) and 1230, 2140, 4020, 7430 and 14700 mg/kg bw/day (females). Also these doses were significantly above the currently established limit doses by OECD and EPA. The final mean body weight and body weight gain of 10,000 ppm females were significantly greater than those of the controls. The 80,000 ppm group female mice experienced toxicity with dehydration stress (40% decrease in water intake), hypoactivity, lethality and pale feces as evidence of this exposure concentration exceeding a MTD in this group of animals. Relative liver weights of 40,000 and 80,000 ppm males and 80,000 ppm females were significantly increased. The estrous cycle of 80,000 ppm females was significantly longer than that of the controls. There were no significant differences in sperm motility parameters between exposed and control males. Similarly to the rat study, the biological relevance of the effects on fertility is considered to be questionable.

 

The longer estrous cycle in the 80,000 ppm group female mice requires additional analysis and discussion. It is well accepted that caloric restrictions in female mice can cause lengthening of estrus cycles (Biology of the Laboratory Mouse 1966; Chapin et al. 1993). Although the feed consumption values were not provided in the NTP study, feed consumption values typically mirror water consumption values in rodent species (Duffy et al. 1989). In addition, the length of estrous cycles in female mice are highly variable, with a general range from 4-5 days (Cooper and Goldman 1998). Specific to the mouse strain used in the NTP study, B6C3F1 female control mice have been reported to have estrous cycle lengths of 4.20 (±0.11), 4.40 (±0.314), 4.45 (±0.30) and 4.95 (±0.42) days in a random sample of NTP 90-day studies (NTP Technical Reports of Methylene Blue Trihydrate (CAS No. 7220-79-3),p-tert-butylcatechol (CAS No. 98-29-3), Formamide (CAS No. 75-12-7) and Bromochloroacetic Acid (CAS No. 5589-96-8))(NTP, 2002, 2006, 2008, 2009). The length of the estrous cycle in the female mice in the 80,000 ppm group was 4.56 days, well within the control group values described above. In contrast, the estrous cycle length in the control group female animals from the NTP study was 4.05 days, considerably shorter than similar NTP 90-day control results. This suggests that the difference in estrous cycle length between the control and 80,000 ppm group in the current NTP study was more likely a result of an aberrant control group value, rather than a true treatment related effect.

 

The lack of animal evidence for targetted reproductive toxicity for dipropylene glycol is supported by the chemistry of this substance. The chemical structure of dipropylene glycol is made up of twooxypropylene unitshaving aliphatic hydrocarbon (-C-C-) and aliphatic ether (-C-O-C-) backbone and two aliphatic alcohol (-C-OH) groups. Dipropylene glycol furtherlacks specific structural features that are associated with nuclear receptor binding affinity of hydrogen bond donor associated with single or multiple hydrophobic aromatic rings such as phenol or aniline (Schmieder et al. 2008). The affinity with which propylene glycol substances, including dipropylene glycol,could bind to the estrogen and androgen receptors was estiamted with the TIssue MEtabolism Simulator (OASIS TIMES software v2.27.5, LMC, Bulgaria) structure-activity relationship algorithm (Westet al., 2014). The TIMES modeling approach is based on a Common Reactivity Pattern (COREPA) which assesses the impact of three-dimensional (3-D) molecular conformation distributions and flexibility on stereo-electronic properties of the modeled substances (Mekenyanet al.2004; Mekenyan and Serafimova 2009). The modeling assessment predicted no activity against both the human estrogen and androgen nuclear receptor binding.  Thus, the modeling findings indicated thatdipropylene glycolhas no potential for endocrine disruptionviadirect receptor binding agonist or antagonist modes of action and this substance should not be considered as a potential endocrine disruptor (West et al. 2014). This conclusion agrees with the assumptions based on the chemical structure ofdipropylene glycol that lacks the necessary motifs associated with specificity for receptor-ligand interactions within the ER binding pocket.  

In the 2 -year drinking water study with rats, exposure concentrations were 0, 2500, 10000 and 40000 ppm, corresponding to actual ingested doses of 115, 470 and 3040 mg/kg bw/day in male and 140, 530 and 2330 mg/kg bw/day in female rats. Again, the dose levels exceeded the currently established limit dose of 1000 mg/kg bw/day according to OECD and EPA guidelines. Survival of 40,000 ppm males was significantly less than that of the control group. Reduced survival was largely due to a high rate of moribund sacrifices that occurred between days 431 and 690; moribundity being probably caused by chronic progressive nephropathy (CPN) and subsequent renal insufficiency. 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 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 expert review of Hard et al (2009), rodent CPN has no strict counterpart in humans, and therefore these changes should be regarded as having no significance for human risk assessment.

The incidences of parathyroid gland hyperplasia and heart mineralization were significantly increased in 40,000 ppm males. These lesions are considered to be secondary to chronic nephropathy.

The incidences of minimal to mild focal granulomatous inflammation of the liver were significantly increased in 10,000 and 40,000 ppm males and slightly increased in 10000 ppm females. This 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. The observed incidences were also comparable with the historical control data for chronic non-neoplastic lesions in F-344 rats, (Experimental Pathology Laboratories, Inc. 2008) and therefore regarded as irrelevant for human risk assessment. The incidences of bile duct hyperplasia in 40,000 ppm males and females were significantly greater than those in the controls. In addition, 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 those in the controls. The incidence of mild to marked thrombosis in males exposed to 40,000 ppm was significantly increased. The biological significance of these nasal lesions is not certain but could be related to metabolism of dipropylene glycol in the olfactory epithelium. Overall, the NOAELs of 470 mg/kg bw/day and 530 mg/kg bw/day were established for male and female rats, respectively, based on the effects in the liver and the increase of nasal lesions.

In the 2 -year study with mice, dose levels of 0, 10000, 20000 and 40000 ppm were chosen, corresponding to actual ingested doses of 735, 1220 and 2390 mg/kg bw/day (males) and 575, 1040 and 1950 mg/kg bw/day (females). Mean body weights of 40,000 ppm male mice were less than those of the controls throughout the study; mean body weights of females exposed to 40,000 ppm were less than those of the controls during the second year of the study. At study termination, mean body weights of male and female mice exposed to 40,000 ppm were approximately 23% and 14% less than those of the controls, respectively. The incidence of hepatocellular adenoma in male mice exposed to 40,000 ppm was significantly less than that in the controls and was at the lower end of the historical range for controls (all routes) given the NTP-2000 diet. This was considered to be related to the decrease in body weight observed in the 40,000 ppm group. The incidences of alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or carcinoma (combined) in 10,000 ppm males were greater than those in the controls; however, these incidences were not exposure concentration related and were within the historical ranges for controls (all routes) given NTP-2000 diet. There were exposure concentration-related decreases in the incidences of focal hyperplasia and focal hypertrophy of the adrenal cortex in exposed males. The incidences of focal hyperplasia in 20,000 and 40,000 ppm males and focal hypertrophy in 40,000 ppm males were significantly less than those in the controls. The incidences of these changes occurred at a variable rate, and it is uncertain if the decreases were exposure related. The NOAELs were 1220 mgkg bw/day for male and 1040 mg/kg bw/day for female mice (20000 ppm), based on the decreased body weights in the highest dose group.

References not listed in the basic data set

 

Biology of the Laboratory Mouse (1966), E.L. Green, Editor, 2nd Edition. Dover Publications, Inc., New York.

 

Blazak WF, Ernst TL and Stweart BE. (1985).Potential Indicators of Reproductive Toxicity: Testicular Sperm Production and Epididymal Sperm Number, Transit Time, and Motility in Fischer 344 Rats. Tox. Sci.5(6:1):1097-1103.

 

Chapin RE, Gulati DK, Barnes LH and Teague JL. (1993). The effects of feed restriction on reproductive function in sprague-Dawley rats.Fundam. Appl. Toxicol. 20:23-29.

 

Cooper RL and Goldman JM. (1998). Vaginal Cytology, in Daston G and Kimmel C.eds.,An Evaluation and Interpretation of Reproductive Endpoints for Human Health Risk Assessment. ILSI Press, Developmental and Reproductive Toxicology Committee, International Life Sciences Institute, Health and Environmental Sciences Institute, Washington D.C., Chapter VI, pages 42-56.

 

Duffy PH, Feuers RJ, Leakey JEA,. Nakamura K, Turturro A and Hart RW.(1989). Effects of chronic caloric consumption on the physiologic variables related to energy metabolism in the Fischer 344 rat. Mech. Ageing Devel. 48:117-133.

 

Hamm TE Jr, Working PK and Raynor TH. (1983).The effect of restricted diet on reproduction in F-344 rats. Lab. Animal Sci. 33:505.

 

Holson JF, Nemec MD, Stump DG, Kaufman LE, Lindstrom P and Varsho BJ. (2006). Significance, Reliability and Interpretation of Developmental and Reproductive Toxicity Study Findings. Chapter 9 inDevelopmental and Reproductive Toxicology – A Practical Approach; 2nd Edition, RD Hood, Editor.

 

Marty MS, Chapin RE, Parks LG and Thorsrud BA. (2006). Development of the Male Reproductive System“ Appendix C-4 inDevelopmental and Reproductive Toxicology – A Practical Approach; 2nd Edition, RD Hood, Editor.

 

Mekenyan O and Serafimova R. (2009). Mechanism based modeling of estrogen receptor binding affinity:

a common reactivity pattern (COREPA) implementation. In: Endocrine disruption modeling. Devillers J (ed), CRC Press LLC, Boca Raton, FL, pp 259–294

 

Mekenyan OG, Dimitrov SD, Pavlov TS and Veith GD. (2004). A systematic approach to stimulating

metabolism in computational toxicology.I. The TIMES heuristic modelling framework. Curr Pharm Des.10:1273–1293

 

NTP (National Toxicology Program). (2002). NTP Technical Report on the Toxicity Studies ofp-tert-Butylcatechol (CAS No. 98-29-3) Administered in Feed in F344/N Rats and B6C3F1 Mice. Editor: JK Dunnick.

 

NTP. (2006). NTP Technical Report on the Toxicity and Carcinogenesis Studies of Methylene Blue Trihydrate (CAS No. 7220-79-3) in F344/N Rats and B6C3F1 Mice. Editor: DW Bristol.

 

NTP. (2008). NTP Technical Report on the Toxicity and Carcinogenesis Studies of Bromochloroacetic Acid (CAS No. 5589-96-8) in F344/N Rats and B6C3F1 Mice. Editor: RL Melnick.

 

NTP (2009). NTP Technical Report on the Toxicity and Carcinogenesis Studies of Formamide (CAS No. 75-12-7) in F344/N Rats and B6C3F1 Mice. Editor: RD Irwin.

 

SchmiederaP,Kolanczyk R, Aladjov H, Hornung M, Tapper M, Denny J and Sheedy,B. (2008).Expert consultation on the use of (quantitative) structure activity relationships [(Q)SAR] of estrogen binding affinity to support prioritization of heterogeneous chemicals within defined inventories for screening and testing. Organization for Economic Cooperation and Development (OECD), Paris, France. 17 Feb. 2008

 

West R, Banton M, Hu J and Klapacz J. (2014). The distribution, fate, and effects of propylene glycol substances in the environment.Reviews of Environmental Contamination and Toxicology.232:107-138.

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

Justification for classification or non-classification

Based on the results of the subchronic and chronic drinking water study with rats (observed NOAEL = 425 mg/kg bw/d in 90 -day study and 470 mg/kg bw/day in 2 -year study, which is above the cut-off value of 50 mg/kg bw/day established for the 90-day study), classification is not warranted according to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.