Fatty acids, C18-36, esters with ethylene glycol

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:

R.A. Corley et al.

Year:

2008

Bibliographic source:

Toxicology and Applied Pharmacology 228 (2008) 165–178

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

Test during 1 year in males only. No haematology or clinical chemistry. No behavioural examinations. The study focusses on effects on the kidney

Principles of method if other than guideline:

Test during 1 year in males only. No haematology or clinical chemistry. No behavioural examinations.
The study focusses on effects on the kidney

GLP compliance:

not specified

Remarks:

publication

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

Details on test material:

- Name of test material (as cited in study report): ethylene glycol
- Molecular formula (if other than submission substance): C2H6O2
- Analytical purity: 99.4%±0.07%

Specific details on test material used for the study:

- Analytical purity: 99.4%±0.07%

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Based on literature suggesting that Wistar rats may be most susceptible to ethylene glycol-induced nephrotoxicity ( and that males may be more sensitive than females)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar Han (Crl:WI(Gix/BRL/Han)IGSBR
- Source: Charles River Laboratories, Inc. (Raleigh, North Carolina)
- Age at study initiation: 6 weeks
- Weight at study initiation: ca 180 g (taken from figure in publication)
- Housing: individually in stainless-steel mesh caging suspended above cageboard
- Diet: lower-protein NTP2000 diet in meal form from Zeigler Brothers, Inc., Gardners, PA ad libitum
- Water: tap water ad libitum
- Acclimation period: no data

DETAILS OF FOOD AND WATER QUALITY: not provided

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5–22.3 °C
- Humidity (%): 48.6–63.0%.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts: serially diluting a concentrated test material-feed mixture (premix) with ground feed, passing it through a Comil (Quadro Engineering Incorporated, Waterloo, Ontario) to break any small clumps, then further mixed with NTP2000 diet using a Hobart blender.
- Storage temperature of food: ambient

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

analysed for homogeneity, stability and accuracy of preparation by a solvent extraction method followed by gas chromatography/mass spectrometry
Found to be stable, no more details available

Duration of treatment / exposure:

1 year

Frequency of treatment:

continuously diet formulation concentrations were adjusted once weekly during the first 12 weeks and two-weekly thereafter

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males/dose (an additional 5 males/ dose for analysis of ethylene glycol, glycolic acid and oxalic acid in blood, kidneys and urine

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on a 16 weeks study with a NOAEL for renal toxicity in both male Wistar and F344 rats at 150 mg/kg/day and severe effects at 500 and 1000 mg/kg bw

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: (bi)-weekly

BODY WEIGHT: Yes
- Time schedule for examinations:weekly (first 12 weeks)/bi -weekly

FOOD CONSUMPTION AND COMPOUND INTAKE : weekly adjusted for first 12 weeks, bi-weekly thereafter
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated based food consumption and body weight data: Yes

FOOD EFFICIENCY: not specified

WATER CONSUMPTION; Yes
- Time schedule for examinations: at study end during over a 24 hour period

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: Yes
- Time schedule for collection of urine: at study end over a 24 hour period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked: Specific gravity, pH, color, and appearance
Semiquantitative analysis of pH, bilirubin, glucose, protein, ketones, occult blood, and urobilinogen
Microscopic evaluation of crystal types via micro-sediment analysis .

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes on all animals not further specified

ORGAN WEIGHTS: Liver and kidney

HISTOPATHOLOGY: Yes on kidneys by light and fluorescence microscopy (half used, with other half used for metabolite analysis) and urinary bladder

Statistics:

Statistical analyses were conducted using SAS/STAT Software Version 6 or Prism 4 for Windows. Means and standard deviations were calculated for all continuous data. Body weights, feed consumption, organ weights, urine volume, and urine specific gravity were evaluated by Bartlett's test for equality of variances (alpha = 0.01; Winer, 1971). Based on the outcome of Bartlett's test, exploratory data analyses were performed by a parametric (Steel and Torrie, 1960) or nonparametric analysis of variance (ANOVA; Hollander and Wolfe, 1973). If the ANOVA was significant at alpha = 0.05, it was followed, sequentially, by Dunnett's test (Winer, 1971) or the Wilcoxon Rank-Sum test (Hollander and Wolfe, 1973) with a Bonferroni correction for multiple comparisons to the control (Miller, 1966). Metabolite levels were evaluated by a one-way analysis of variance (with log transformation of the data) followed by Dunnett's test to compare each treatment group to controls.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at 300 and 400 mg/kg bw not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

1 male at 0 and 150 mg/kg bw (non-treatment related, spontaneous lymphoid tumor)
5 males at 300 mg/kg bw ( found dead or killed in extremis)
4 males at 500 mg/kg bw before day 203 (found dead or killed in extremis): 16 males at 400 mg/kg bw (killed in extremis on day 203)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at 400 mg/kg bw excessive body weight loss (therefore animals were terminated on day 203)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

at 400 mg/kg bw: sign decreased food consumption

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

increased at 300 mg/kg bw (150% of controls)

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

at 300 mg/kg bw:increased pH (related to metabolic products); increased volume and concomitantly decreased specific gravity compared to controls

Increased calcium oxalate crystals in the urine of the 50, 150, and 300 mg/kg/day groups.

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):

at 300 and 4000 mg/kg bw increased absolute and relative kidney weights (DR)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

at 300 mg/kg bw: calculi in the bladder and sometimes the renal pelvis or ureter in 8 of the total 15 rats (in non survivors hemorrhage of the bladder wall, usually with ascites or other edematous changes)
at 400 mg/kg bw: calculi in the bladder and sometimes the renal pelvis or ureter in 8 of the total 20 rats, a roughened kidney surface, renal pelvic dilatation, thickened bladder wall (early decedents hemorrhage of the bladder wall)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

at 300 mg/kg bw : nephropathy associated with crystal deposition in most animals
at 400 mg/kg bw : nephropathy associated with crystal deposition in all animals

Light microscopy
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 foamy, 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 intralumenal neutrophils, dilatation of the renal pelvis, and some transitional cell hyperplasia of the renal pelvic lining.

Fluoresence microscopy
Birefringent, polycrystalline particles arranged in rosette, fan-shaped or sheaf-like patterns, or individually as near-rectangular plates, were observed in 8 of 13 rats receiving 300 mg/kg/day, and in 10 of 10 rats receiving 400 mg/kg/day. Depending on the severity, crystal deposition occurred in the lumens of tubules from the cortex to the papilla, in outpouchings of the papilla lining, and in the renal pelvis, particularly in the fornices.

Other effects:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

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

Applicant's summary and conclusion

Conclusions:

The NOAEL for kidney effects from this study was 150 mg/kg bw

Executive summary:

A chronic toxicity study was conducted in male Wistar rats administered ethylene glycol via the diet at 0, 50, 150, 300, or 400 mg/kg/day for up to twelve months. Mortality occurred in 5 of 20 rats at 300 mg/kg/day (days 111–221) and 4 of 20 rats at 400 mg/kg/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/day presumably due to osmotic diuresis. Calculi (calcium oxalate crystals) occurred in the bladder or renal pelvis at ≥300 mg/kg bw.

Rats dying early at ≥300 mg/kg/day had transitional cell hyperplasia with inflammation and hemorrhage 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/day, all at 400 mg/kg/day, but none at ≤150 mg/kg/day.

The NOAEL of 150 mg/kg/day 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.