Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Oral:
A NOEL of 150 mg/kg bw/day was determined in rats with regard to oral exposure. The kidneys were found to be the target organ at higher doses.
Dermal:
A NOAEL was found to be 2200 mg/kg bw/day in dogs.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Wistar and Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age: appr. 26 days
- Housing: individually
- Animals were maintained in accordance with the "Guide for the Care and Use of Laboratory Animals".
- Acclimation period: 20 days
Route of administration:
oral: feed
Details on oral exposure:
For each mixture, the needed amount of test material was weighed and mixed into an appropriate amount of NTP2000 diet (Ziegler Bros, Inc.) using a Hobart blender. The test diets were prepared weekly and stored under ambient conditions. Dietary concentrations were adjusted weekly based on group mean body weights and feed consumption for each group of rats.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of test material in the diets were confirmed by gas chromatography during weeks 0, 1, 2, 3, 4, 8, and 15.
Duration of treatment / exposure:
16 weeks
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 F344, 10 Wistar rats
Control animals:
yes, plain diet
Details on study design:
Groups of 10 male F-344 and Wistar rats received the test material via the diet at doses of 0, 50, 150, 500, or 1000 mg/kg bw/day for 16 weeks. Dietary concentrations were adjusted weekly based on group mean body weights and feed consumption for each group of rats. At the end of 16 weeks of exposure, each animal received a complete necropsy with kidneys evaluated by histopathology.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS: Hematological, urine, and clinical chemistry analyses had been conducted in prior subchronic and chronic studies. Only limited urinalyses were conducted in this study as a complement to the renal pathology examinations. Urine was collected over an approximately 24-h period prior to necropsy for all animals (fasted) using metabolism cages. The following parameters were evaluated: specific gravity, pH, color, appearance, protein, glucose, bilirubin, urobilinogen, ketones, occult blood, leukocytes, nitrites, total volume, color, appearance, and microscopy of sediment.


Sacrifice and pathology:
After 16 weeks of treatment, all surviving animals were euthanized. Selected tissues (44/animal) were preserved in neutral buffered formalin for potential future evaluations. The kidneys were weighed and the ratio to the terminal body weight was calculated for each animal. Sections of each kidney (one longitudinal in a mid-sagittal position and from the other kidney, a transverse section through the renal papilla) were imbedded in paraffin, processed, and cut at 5 mm thickness, mounted on glass microscopic slides, and stained with hematoxylineosin. The slides were examined by normal light (brightfield) for pathologic lesions, by polarized light for the presence of oxalate crystals and by fluorescence microscopy for the presence of lysosomes. The severity of compound-induced nephropathy was determined and crystal deposition was measured.
Other examinations:
Additionally, toxikokinetics parameters were examined. Satellite groups of five rats/strain/dose level received the test material via diet at 0, 150, 500, and 1000 mg/kg bw/day for either 1 week or 16 weeks of exposure. For 24 h prior to euthanasia, urine was collected while the animals were housed separately in metabolism cages. Prior to euthanasia a blood sample was obtained. At euthanasia, the kidneys were removed. Blood and urine samples were analyzed for ethylene glycol, glycolic acid, and oxalic acid by gas chromatography/mass spectrometry (GC/MS). Kidneys were first homogenized directly (no diluent) then analyzed by the method used for analysis of blood. For details and results, please refer to IUCLID5, section 7.1.
Statistics:
Body weight, body weight change, feed consumption, urinalysis, and kidney weight data were subjected to a parametric one-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistical significance ( p 5 0.05), Dunnett’s test was used to compare the test article-treated groups to the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
Besides two Wistar rats of the 1000 mg/kg bw/day dose group dying in week 15, no other deaths occured and no clinical signs were observed.
Mortality:
mortality observed, treatment-related
Description (incidence):
Besides two Wistar rats of the 1000 mg/kg bw/day dose group dying in week 15, no other deaths occured and no clinical signs were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Only Wistar rats treated with 500 and 1000 mg/kg bw/day exhibited decreased mean body weights.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg bw/day groups of both strains reduced food consumption was observed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
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 were significantly higher in the 1000 mg/kg bw/day F-344 and in the 500 and 1000 mg/kg bw/day Wistar rat groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Crystal deposition in the kidneys was observed in the in the 500 and 1000 mg/kg bw/day Wistar rat groups. Crystal deposition was also observed in the 1000 mg/kg bw/day group in the F-344 strain, but to a lesser extent.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Nephropathy was observed in the kidneys in the 500 and 1000 mg/kg bw/day Wistar rat groups, associated with crystal deposition.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Key result
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
System:
other: urogenital
Organ:
kidney
Treatment related:
yes
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Available data are considered as reliable.
System:
other: urogenital
Organ:
kidney

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

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
Principles of method if other than guideline:
In a previous study (BASF 80S0385/8243), one male tested with 2 mL/kg bw of the test item showed a testicular atrophy and a diffuse impairment of spermatogenesis. In the group of animals treated with 8 mL/kg bw, the tested concentration was clearly toxic and testicular atrophy with diffuse impairment of spermatogenesis was seen in all treated animals. A further study (BASF 60D0182/8507) was conducted to clarify whether the testicular findings reported above were related to oxalate nephrosis or due to the p-tert.-butyl benzoate present in the test item. For this purpose, one group of animals was treated with 4 mL/kg bw of ethylene glycol with inhibitors (=< 91.8% ethylene glycol, >= 2.8% p-tert.-butyl benzoate) and a further group was treated with ethylene glycol (purity > 99.5%). In this study the 4-week dermal application of both monoethylene glycol and Glysantin G 105 (ethylene glycol with inhibitors) at a dose level of 4.0 mL/kg bw led to erythema on the application area varying in terms of degree and time. However, erythema should be assessed as a questionable test substance effect of subordinate importance since it was only observed on the application area and it was not possible to make a definite differentiation between an effect resulting from the application procedure and a finding relevant for the substance. The slight impairments of spermatogenesis and spermiogenesis observed in individual male dogs of the group treated with 4 mL/kg bw Glysantin G 105 and the sperm content in the epididymis that was reduced compared with the 2 other test groups were however assessed as a definite substance-induced effect. Although these changes were less pronounced than in the group treated with 8 mL/kg bw Glysantin G 105 (BASF 80S0385/8243) and the animals of test group treated with 4 mL/kg bw of ethylene glycol showed no differences from the control group in this study, it was assumed that p-tert.-butyl benzoate contained in Glysantin G 105 might have been responsible of the testicular damage. To verify this assumption, a further study was undertaken, testing Glysantine G 105 (without increased content of p-tert.-butyl benzoate) at dose levels of 2 and 4 mL/kg bw.

GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
purity: > 93.4% ethylene glycol and < 1.3% p-tert.butylbenzoic acid
Species:
dog
Strain:
Beagle
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: about 14 - 15 months
- Weight at study initiation: 11.3 kg
- Fasting period before study: before blood and urine collection as well as before necropsy, a fasting period of 16 hours was warranted
- Housing: singly in kennels
- Diet: pelleted diet, daily 400 g
- Water: drinking water, ad libitum
- Acclimation period: 7 days
Type of coverage:
not specified
Vehicle:
unchanged (no vehicle)
Details on exposure:
Before application period, the dogs were shaved on the back, flanks, front and waist. The undiluted test substance was applied daily onto the clipped skin. The application area was about 60% of the total body surface. The test substance was not washed off. All animals including those of the control group wore rigid plastic collars to prevent any ingestion of the test substance. Dermal application was carried out up to the day of the specific necropsy of the animals.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily
Dose / conc.:
2 other: mL/kg bw
Dose / conc.:
4 other: mL/kg bw
No. of animals per sex per dose:
4
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
Check of dead animals twice a day.
Clinical symptoms twice a day.
Daily food consumption and body weight determination.
Sacrifice and pathology:
All animals were assessed by gross pathology, and then a histopathological examination was carried out.
Other examinations:
Two urinalyses were carried out.
Statistics:
yes
Clinical signs:
no effects observed
Dermal irritation:
not examined
Mortality:
no mortality observed
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Incidence of calcium oxalate crystals in the urine of 3 of the 4 male dogs varying in terms of degree and time.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Several birefringent precipitates in the kidney of one male dog, some of them showing a positive calcium oxalate reaction.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Several birefringent precipitates in the kidney of one male dog, some of them showing a positive calcium oxalate reaction.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
2 mL/kg bw:
There were no definite changes that might be causally related to the test substance applied in any of the examinations carried out.
Key result
Dose descriptor:
NOAEL
Effect level:
> 2 200 - < 4 400 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
2 other: mL/kg bw
System:
other: urogenital
Organ:
kidney
Treatment related:
yes
Conclusions:
No testicular damage that was definitely induced by the test substance was detected in any of the male dogs investigated.
LD50 dermal (dog): > 4000 mg/kg bw
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
2 200 mg/kg bw/day
Study duration:
subacute
Species:
dog
Quality of whole database:
GLP and guideline study
System:
other: urogenital
Organ:
kidney

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated oral toxicity:

Schladt et al. (1998) tested ethylene glycol in an oral (gavage) study. Male and female rats were given doses of 220, 660 and 2000 mg/kg over a study period of 33 days. The daily application resulted in treatment-related effects on the kidneys. A slight derease in the urinary excretion of potassium, calcium and phosphate (males), a diminished pH value of the urine, and a slight increase in osmolality (females) were observed. In both sexes, excretion of oxalate was significantly increased and microscopic examination of urinary sediment revealed calcium oxalate crystals. Kidney weights of males and females were slightly elevated. Histopathology revealed crystals in renal tubuli, renal pelvis, and urinary bladder; tubulopathy and epithelial hyperplasia within the renal pelvis were also observed. The NOAEL could not be clearly determined, probably 220 mg/kg.

In a publication (Cruzan et al. 2004), a subchronic toxicity study with ethylene glycol is described which was carried out in F-344 and Wistar rats for comparison. A NOEL of 150 mg/kg bw/day was revealed for both strains. It is also described in the publication that the different strains of rats may show different susceptibilities in terms of renal pathology:A higher sensitivity of male Wistar rats than male Fischer rats towards chronic MEG-induced nephrotoxicity was reported in the frame of the 16 weeks experiment in which the animals received dietary doses of 0, 50, 150, 500 and 1000 mg/kg bw and day. The incidence and severity of the lesions was strongly related to the accumulation of oxalate crystals in the kidney (see chapter Toxicokinetics).

Corley et al. (2008) offered dietary doses of 0, 50, 150, 300 or 400 mg/kg for up to 12 months. The authors concluded that all rat NOAELs appear to converge at an overall NOAEL of 150 mg/kg for rats and there is a threshold dose below which no renal toxicity occurs, regardless of exposure duration. Furthermore, the authors pointed out that other species like dogs and humans have higher clearance ratios for oxalic acid than rats.

NTP (1993) reported an oral feeding study with mice. Animals were given concentrations of 3200, 6300, 12500, 25000 and 50000 ppm for 13 weeks. All animals survived to the end of the study and final mean body weight of exposed mice did not differ significantly from those of the controls. Only male mice in the 25000 and 50000 ppm groups exhibited histopathologic lesions (nephropathy and hepatocellular hyaline degeneration). The NOAEL was found to be 12500 ppm.

Repeated inhalation toxicity:

No formal valid studies are available. However, the inhalation toxicity is assessable from the inhalation studies on prenatal toxicity (Tyl et al. 1995; see chapter 7.8.2). These studies show that 500 mg/m3 (nose-only) or 150 mg/m3 (whole-body) did not affect the kidneys which are the classical target organ for MEG. It is pointed out that chamber exposure to MEG aerosols leads also to an oral uptake from the fur which in a proportion which is difficult to quantify.

Repeated dermal toxicity:

BASF (1991) reported a 4-week dermal application study according to OECD guideline 410. Male dogs were given the unchanged test substance (> 92.5% ethylene glycol and < 1.42% na-p-tert-butyl-benzoat; PTBBA) daily to the clipped skin in concentrations of 0.5, 2 and 8 ml/kg. Further studies followed to establish whether the severe testicular damage that was found in all dogs at 8 ml/kg and in one dog at 2 ml/kg was caused by na-p-tert-butyl-benzoic acid or whether it must be regarded as a direct or indirect consequence of ethylene glycol administration. Some urinary oxalate crystals were found at 2 ml/kg but due to the absence of histological findings considered as not adverse. The NOEL was 2 ml/kg b.w . (2220 mg/kg b.w.). At this level, some urinary oxalate crystals were observed but considered as not adverse due to the absence of histological findings.

BASF (1991) reported an additional 4-week dermal application study according to OECD guideline 410. In this study, male dogs received the unchanged test substance (> 93.4% ethylene glycol and < 1.3% p-tert.butylbenzoic acid) daily to the clipped skin in amounts of 2 and 4 ml/kg. No testicular damage that was definitely induced by the test substance was detected in any of the dogs investigated. The NOEL was found to be 4 ml/kg b.w. (4440 mg/kg b.w.). At this level, urinary oxalate crystals were observed but considered as not adverse due to the absence of histological findings.

BASF (1991) reported a supplementary 4-week dermal application study according to OECD guideline 410. In this study, male dogs were given the unchanged test substance (> 91.8% ethylene glycol < 2.8% p-tert.butylbenzoic acid) daily to the clipped skin in a concentration of 4 ml/kg. A slight impairment of spermatogenesis and spermiogenesis was observed in individual male dogs of the test group and the sperm content in the epididymis was reduced. Although, these changes were less pronounced than in the 8 ml/kg group of the first study and the animals of test group 4 ml/kg showed no differences from the control group in this study, it is assumed that the p-tert.butylbenzoic acid content in the test substance has caused the testicular damage (see attachment: Hunter et al., 1965: Studies on the Oral Toxicity of p-tert-butyl Benzoic Acid in Rats. Fd. Cosmet. Toxicol. 3, 289 - 298). A NOEL was not defined due to dermal erythema.

Tyl et al. (1995) reported about the assessment of the developmental toxicity of ethylene glycol applied cutaneously to mice. No clear adverse effects were found in maternal animals dermally exposed to EG at 3549 mg/kg. This approximate NOAEL in mice supports the NOAEL obtained from the dog studies.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is considered to be classified and labelled with STOT RE Cat. 2, H373 (May cause damage to organs) for repeated dose toxicity (oral route) under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218.

Classification concerning repeated dermal and repeated inhalation toxicity is not warranted.