Reaction mass of ethane-1,2-diol and 2-hydroxyethyl formate and ethylene diformate

Repeated dose toxicity: oral

Administrative data

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Dosimetry considerations in the enhanced sensitivity of male Wistar rats to chronic ethylene glycol-induced nephrotoxicity.

Author:

Corley RA, Wilson DM, Hard GC, Stebbins KE, Bartels MJ, Soelberg JJ, Dryzga MD, Gingell R, McMartin KE, Snellings WM

Year:

2008

Bibliographic source:

Toxicol Appl Pharmacol 228: 165-178

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

limited number of examined parameters

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethane-1,2-diol

EC Number:

203-473-3

EC Name:

Ethane-1,2-diol

Cas Number:

107-21-1

Molecular formula:

C2H6O2

IUPAC Name:

ethylene glycol

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The male Wistar rat is the most sensitive animal model regarding ethane-1,2-diol toxicity (Corley et al., Toxicol and Appl Pharmacol. (2008), 165-178).

Sex:

male

Details on test animals or test system and environmental conditions:

Male Wistar Han (Crl:\Vl(Gi.x/BRL/Han)lGSBR were obtained from Charles River Laboratories, Inc. (Raleigh, North Carolina). Animals were housed individually in stainless-steel mesh caging suspended above cageboard. Drinking water was available ad libidum except when water consumption was determined from water bottles at the end of the study. Room temperature was maintained within a range of 21.5-22.3 °C and relative humidity within a range of 48.6- 63.0%. Light timers were set to provide a 12-hour light/12-hour dark photoperiod. Animals were fed lower-protein NTP2000 diet, ad libitum. Animals were maintained in accordance with the Guide for the Care and use of Laboratory Animals (1996).

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Up to 12 months

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Plain diet

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 5

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 males/dose

Control animals:

yes, plain diet

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related clinical signs at 50 or 150 mg/kg bw/day. The clincial signs observed at higher doses are not specified in the publication.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality occurred in 5 of 20 rats at 300 mg/kg bw/day (days 111-221) and 4 of 20 rats at 400 mg/kg bw/day (days 43-193), with remaining rats at this dose euthanized early (day 203) due to excessive weight loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was excessive body weight loss at 400 mg/kg bw/day. Body weights for rats given 300 mg/kg bw/day were typically lower but not statistically lower than controls by mid-study, There were no body weight effects at 50 or 150 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Rats given 400 mg/kg bw/day had treatment-related decreases in feed consumption at every time point through termination on day 203. There were no treatment related
effects on feed consumption for rats given <= 300 mg/kg bw/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Increased water consumption occurred at 300 mg/kg bw/day.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Increased urine volume with decreased specific gravity occurred at 300 mg/kg bw/day

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean absolute and relative kidney weights in animals given 300 or 400 mg/kg bw/day appeared to be higher than the control group at necropsy. These increases were not statistically significant at 300 mg/kg bw/day and were not statistically analyzed at 400 mg/kg bw/day due to the lack of concurrent controls for the early termination o this dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related gross observations occurred in animals given 300 or 400 mg/kg bw/day and were primarily confined to the kidney and urinary bladder, with secondary treatment-related observations occurring in the lung. The most relevant observation in the 300 mg/kg bw/day group was the presence of calculi in the bladder (and sometimes in the renal pelvis or ureter) in 8 of the total 15 rats examined. This also occurred in 8 of 20 rats at 400 mg/kg bw/day. Calculus formation in the urinary bladder was usually accompanied by dilatation of the bladder and, for the 5 unscheduled deaths at 300 mg/kg bw/day, hemorrhage of the bladder wall, usually with ascites or other edematous changes. Three animals given 300 mg/kg bw/day had calculi in the renal pelvis. Almost all rats at 400 mg/kg bw/day showed gross signs of kidney and/or urinary bladder involvement, including a roughened kidney surface, renal pelvic dilatation, thickened bladder wall, and calculi in the renal pelvis, ureter, or bladder. Of the four unscheduled deaths occurring before early termination of this group, three were observed to have hemorrhage of the bladder wall. All other gross pathological findings were considered secondary to effects observed in the kidneys and bladder, significant body weight loss (400 mg/kg bw/day), or agonal changes prior to death.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Examination by brightfield microscopy showed that a compound-induced nephropathy associated with crystal deposition affected the majority of the animals at 300 mg/kg bw/day and all of those given 400 mg/kg bw/day. Nephropathy was observed as foci, radial tracts, or diffuse areas of basophilic tubules in the cortex, and outer
and inner medulla. The cytoplasm of basophilic proximal tubule cells was loamy, finely vacuolated, or rarefied, with an occasional apoptotic cell or mitotic figure, and mild basement membrane thickening. There was minimal to mild mononuclear inflammatory infiltration and fibrosis accompanying the basophilic alteration. Increasing severity of the nephropathy was manifested by coalescence of foci into areas of diffuse change and an association with tubule dilatation, increasing fibrosis, increasing extracellular matrix, minor tubulitis associated with intraluminal neutrophils, dilatation of the renal pelvis, and some transitional cell hyperplasia of the renal pelvic lining. Proximal tubule mineralization was seen in a few advanced cases, but this was not a constant feature. In many kidneys with compound-induced nephropathy, the outlines of crystals could be observed within tubule lumens, or in the renal pelvis, but these were better visualized and scored for severity
under polarized light optics. A few rats at the highest dose had either minimal degeneration of the papilla tip, or some pyelitis, both associated with crystal deposition. None of the renal alterations associated with ethylene glycol exposure was observed in the rats given 50 or 150 mg/kg bw/day, establishing the Iatter dose as a NOAEL.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Increased water consumption and urine volume with decreased specific gravity occurred at 300 mg/kg bw/day presumably due to osmotic diuresis. Calculi (calcium oxalate crystals) occurred in the bladder or renal pelvis at > = 300 mg/kg bw/day. Rats dying early at > = 300 mg/kg bw/day had transitional cell hyperplasia with inflammation and haemorrhage of the bladder wall. Crystal nephropathy (basophilic foci, tubule or pelvic dilatation, birefringent crystals in the pelvic fornix, or transitional cell hyperplasia) affected most rats at 300 mg/kg bw/day, all at 400 mg/kg bw/day, but none at < =150 mg/kg bw/day. No significant differences in kidney oxalate levels, the metabolite responsible for renal toxicity, were observed among control, 50 and 150 mg/kg bw/day groups. At 300 and 400 mg/kg bw/day, oxalate levels increased proportionally with the nephrotoxicity score supporting the oxalate crystal-induced nephrotoxicity mode of action. No treatment-related effects on the renal clearance of intravenously infused (3)H-inulin, a marker for glomerular filtration, and (14)C-oxalic acid were observed in rats surviving 12 months of exposure to ethylene glycol up to 300 mg/kg bw/day. In studies with naïve male Wistar and F344 rats (a less sensitive strain), a significant difference was observed in oxalate clearances between young rats (i.e. Wistar clearance < F344) but not in age-matched old rats. Regardless, the ratios of oxalate:inulin clearances in these two strains of rats, including those exposed to ethylene glycol, were all < 1, suggesting that a fraction of the filtered oxalate is reabsorbed. Other species, including humans, typically have clearance ratios >1 and are more effective at clearing oxalic acid by both glomerular filtration and active secretion. Thus, the lower renal clearance and kidney accumulation of oxalates in male Wistar rats enhances their sensitivity, which will be a factor in human risk assessments. The benchmark dose values (BMD05, BMDL05) were 170 mg/kg bw/day and 150 mg/kg bw/day for nephropathy, and 170 mg/kg bw/day and 160 mg/kg bw/day for birefringent crystals, using incidence times severity data in each case.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

urinary

Organ:

bladder

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

The NOAEL of 150 mg/kg bw/d is the same as that reported after 16-week exposure and appears to be a threshold dose below which no renal toxicity occurs, regardless of exposure duration.

Applicant's summary and conclusion

Conclusions:

A NOAEL of 150 mg/kg bw/d was derived based on the absence of renal toxicity.

Executive summary:

Male Wistar rats were administered ethylene-1,2-diol via the diet at 0, 50, 150, 300, or 400 mg/kg bw/day for up to twelve months. Subgroups of animals were included for metabolite analysis and renal clearance. Mortality occurred in 5 of 20 rats at 300 mg/kg bw/day (days 111-221) and 4 of 20 rats at 400 mg/kg bw/day (days 43-193), with remaining rats at this dose euthanized early (day 203) due to excessive weight loss. Increased water consumption and urine volume with decreased specific gravity occurred at 300 mg/kg bw/day presumably due to osmotic diuresis. Calculi (calcium Oxalate crystals) occurred in the bladder or renal pelvis at >300 mg/kg bw/day. Rats dying early at >300 mg/kg bw/day had transitional cell hyperplasia with inflammation and hemorrhage of the bladder wall. Crystal nephropathy affected most rats at 300 mg/kg bw/day, all at 400 mg/kg bw/day, but none at <= 150 mg/kg bw/day. No significant differences in kidney oxalate levels, the metabolite responsible for renal toxicity, were observed among control, 50 and 150 mg/kg bw/day groups. At 300 and 400 mg/kg bw/day, oxalate levels increased proportionally with the nephrotoxicity score supporting the oxalate crystal-induced nephrotoxicity mode of action. A NOAEL of 150 mg/kg bw/day was determined.