Ethylene carbonate

Endpoint summary

Description of key information

Repeated dose toxicity: oral: No reliable data are available on repeated dose toxicity via the oral route for ethylene carbonate. Data with the read-across substance ethylene glycol, an ethylene carbinate metabolite, were used in a weight of evidence approach to cover the endpoint information requirements. Three reliable repeated dose toxicity studies are available for the oral route. A chronic NOAEL of 150 mg/kg bw/day (actual ingested) was observed in male Wistar rats after 12 months of daily exposure to ethylene glycol. The study was performed according to a guideline equivalent to OECD Guideline 452. In a 10 -day and 90 -day repeated dose toxicity study with ethylene glycol in male and female Sprague-Dawley rats a NOAEL of 1108 mg/kg bw/day (males)/2216 mg/kg/day (females), and a NOAEL of 554 mg/kg bw/day (males)/LOAEL of 554 mg/kg bw/day (females) were determined respectively.

Repeated dose toxicity: dermal and inhalation: No reliable data are available on repeated dose toxicity via the dermal and inhalation route. Reliable chronic toxicity data (12 months exposure) are available from a study with ethylene glycol via the oral route. According to the REACH Regulation, a short-term and sub-chronic repeated dose toxicity test does not need to be conducted if a reliable chronic study is available, provided that an appropriate species and route of administration were used (Column 2 adaptation, Annexes VIII and IX respectively).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 452 (Chronic Toxicity Studies)

GLP compliance:

not specified

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: feed

Duration of treatment / exposure:

12 months

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

20

Control animals:

yes, concurrent no treatment

Clinical signs:

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

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

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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

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

Description (incidence and severity):

Increased water consumption occurred at 300 mg/kg/day presumably due to osmotic diuresis.

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/day presumably due to osmotic diuresis.
No significant differences in kidney oxalate levels, the metabolite responsible for renal toxicity, were observed among control, 50 and 150 mg/kg/day groups. At 300 and 400 mg/kg/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/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/day and 150 mg/kg/day for nephropathy, and 170 mg/kg/day and 160 mg/kg/day for birefringent crystals, using incidence times severity data in each case.

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):

Calculi (calcium oxalate crystals) occurred in the bladder or renal pelvis at > or =300 mg/kg/day. Rats dying early at > or =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 < or =150 mg/kg/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: 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.

Critical effects observed:

not specified

Conclusions:

A chronic NOAEL of 150 mg/kg bw/day (actual ingested) was observed in male Wistar rats after 12 months of daily exposure to ethylene glycol. According to the CLP regulation, substances can be classified as Category 2 for STOT RE if toxic effects with relevance to human health have been observed. Hence, based on responsible care, ethylene glycol and ethylene carbonate are classified as STOT RE category 2, H373 according to the criteria of the CLP Regulation.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The substances discussed in the analogue approach are ethylene carbonate and ethylene glycol, where ethylene carbonate (target substance) is rapidly metabolised to ethylene glycol (source substance) and carbon dioxide. The validity of the proposed read-across is further strengthened by the similarity in the toxicological profiles of both substances. The detailed justification for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Details on results:

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 > or =300 mg/kg/day. Rats dying early at > or =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 < or =150 mg/kg/day. No significant differences in kidney oxalate levels, the metabolite responsible for renal toxicity, were observed among control, 50 and 150 mg/kg/day groups. At 300 and 400 mg/kg/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/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/day and 150 mg/kg/day for nephropathy, and 170 mg/kg/day and 160 mg/kg/day for birefringent crystals, using incidence times severity data in each case.

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male

Basis for effect level:

other: 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

Critical effects observed:

not specified

Conclusions:

A chronic NOAEL of 150 mg/kg bw/day (actual ingested) was observed in male Wistar rats after 12 months of daily exposure to ethylene glycol. According to the CLP regulation, substances can be classified as Category 2 for STOT RE if toxic effects with relevance to human health have been observed. Hence, based on responsible care, ethylene glycol and ethylene carbonate are classified as STOT RE category 2, H373 according to the criteria of the CLP Regulation.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

- Principle of test:
Ethylene glycol was administered in drinking water at concentrations of 0.5, 1.0, 2.0, and 4.0% for both sexes in the 10-day study.
- Short description of test conditions: Animals were housed, two per cage, in an animal room maintained at 21-24°C and 40-60% relative humidity with 12 hour light-dark cycles. Rodent Chow N. 5001 (Ralston Purina Co., St. Louis, MO) and treatment solutions were available ad libitum.
- Parameters analysed / observed: At time of sacrifice necropsies were performed and tissues prepared for histological evaluation. Blood samples were taken for hematology and clinical chemistry determinations. Body weights were measured weekly. Water and food consumption were determined three times weekly.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2-852019
- EG was administered via drinking water solutions which were prepared weekly by the addition of EG to distilled water. The solutions were delivered in amber colored glass bottles with rubber stoppers and stainess steel sipper tubes with ball bearings to avoid loss of test material. The concentrations of solutions administered to the animals was confirmed and the stability of EG in the drinking water was verified.

OTHER SPECIFICS: The chemical was analyzed for purity using gas chromatography and its composition confirmed by mass spectrometry. There were no impurities present within the detectable limits of the instrumentation.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 85 days
- Weight at study initiation: average of 350 grams (males) and 245 grams (females)
- Fasting period before study: no data
- Housing: plastic cages with hardwood bedding (Ab-Sorb-Dri, Inc., Maywood, NJ), two animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week quarantine

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 °C
- Humidity (%): 40-60% relative humidity
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12hr/12hr

IN-LIFE DATES: not specified

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test item solutions were prepared weekly by the addition of EG to distilled water. The solutions were delivered in amber coloredglass bottles with rubber stoppers and stainless steel sipper tubes with ball bearing to avoid loss of test material.
Concentrations of solutions administered: 0, 0.5, 1.0, 2.0, 4.0% EG solution

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): drinking water (distilled water)
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): distilled water was chosen as vehicle. The stability of Ethylene Glycol in the drinking water was verified.
- Concentration in vehicle: 0, 0.5, 1.0, 2.0, 4.0% EG solution
- Amount of vehicle (if gavage): treatment solutions were available ad libitum

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

10 days

Frequency of treatment:

Treatment solution (drinking water) was available ad libitum during the exposure duration. Water bottles were changed on Monday, Wednesday and Friday of each week and consumption was measured at each change.

Dose / conc.:

0 other: % (v/v)

Dose / conc.:

0.5 other: % (v/v)

Remarks:

Theoretical dose: 554 mg/kg/day

Dose / conc.:

1 other: % (v/v)

Remarks:

Theoretical dose: 1108 mg/kg/day

Dose / conc.:

2 other: % (v/v)

Remarks:

Theoretical dose: 2216 mg/kg/day

Dose / conc.:

4 other: % (v/v)

Remarks:

Theoretical dose: 4432 mg/kg/day

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data

Observations and examinations performed and frequency:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for clinical signs of changes in health

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed on the first day of dosing and weekly thereafter. Terminal body weights were measured at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water bottles were changed on Monday, Wednesday and Friday of each week and consumption was measured at each change.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice (via cardiac puncture)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: Hemoglobin, hematocrit, erythrocytes and leukocyte measurements were made with a Model ZBI Coulter Counter

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice (via cardiac puncture)
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: AST, ALT, LDH, cholesterol, phosphorus, calcium, glucose, BUN and creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Complete postmortem examinations were completed on all animals unless they were severely autolyzed.

HISTOPATHOLOGY: Yes
Tissues were fixed in 10% neutral buffered formalin and later trimmed, processed, embedded in paraffin, sectioned and stained with hematoxylin and eosin.
The following tissues were examined for histological changes from all animals in the control group and all animals in highest dose group having at least 60% of the animals surviving until terminal sacrifice: skin, mandibular and mesenteric lymph nodes, mammary glands, thigh muscle, sciatic nerve, femur and bone marrow, thymus, trachea, lungs, duodenum, jejunum, salivary gland, ileum, colon, cecum, rectum, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles, prostate, testes, epididymides, ovaries, uterus, nasal cavity and nasal turbinates, brain, pituitary, preputial and clitoral glands, Zymbal’s gland and any gross lesions. The target organ and any gross lesions were examined histologically from all remaining animals that survived until the terminal sacrifice. Lungs and any gross lesions were examined from all animals that died prior to the terminal sacrifice.

Other examinations:

Organ weights (heart, kidney, liver, lung, spleen, brain, gonads and thymus) were measured at necropsy

Statistics:

Tukey's multiple comparison procedure was used for body weights, organ weights and organ/body weight ratio data. Kruskal-Wallis Rank Sum was used for the clinical chemistry and hematology data. Overall analyses were done by one factor analysis of variance. Values which differed from the vehicle control group at p<0.05 were considered significant. Statistical evaluation of the incidence of renal histopathological lesions was performed using Fisher's Exact Test. The significance of the severity of the lesions was determined by Pearson's Correlation Co-efficient and Correlation Analysis.

Clinical signs:

not specified

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the 10-day study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The only significant effect that ethylene glycol had on body weight was in the high dose male group animals which lost an average of 45 grams (p <0.05) over the 10 days whereas control animals gained an average of 9 grams during that time.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not specified

Description (incidence and severity):

There was a general trend of partiality to ethylene glycol throughout all groups in both males and females.
The concentrations administered (0.5, 1.0, 2.0 and 4.0%) were selected to give theoretical doses of 554, 1108, 2216, and 4432 mg/kg/day, respectively, or 10, 20, 40 and 80 mM/kg/day, based on projected water consumption of 100 ml/kg/day. The rate of consumption was higher than anticipated and the actual doses ranged from 117% (0.5% group) to 121% (1.0% group) of theoretical dose in the males and from 133% (2.0% group) to 165% (4.0% group) in the females.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The only significant hematological results noted in the males were in the 1.0% group where leukocyte counts (12.5 ± 2.4 x 1E03/µL) were significantly elevated. Leukocyte levels were also elevated, compared to control values (9.1 ± 2.6 x 1E03/µL), in all other exposure groups of males, but not significantly.
Females demonstrated significantly decreased levels for hemoglobin, hematocrit, erythrocytes, and leukocytes in the 4.0% group.
These values over their corresponding control values were: 14.0 ± 0.7/15.1 ± 0.9 g/dL, 35.8 ± 1.9/39.3 ± 2.8%, 6.5 ± 0.4/7.1 ± 0.3 x 1E06/µL, and 6.0 ± 1.6/9.2 ± 2.7 x 1E03/µL.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the male rats there were significant decreases in AST and LDH levels for all test groups when compared to vehicle control and LDH values decreased in a dose response manner. ALT was unaffected, Cholesterol level in the 0.5% group was significantly below that of controls. Calcium, BUN and creatinine for the 4.0% group were significantly above control levels. In addition, creatinine levels in the 1.0% group were significantly above control levels. More varying effects were seen in the females. LDH was not affected, but significant decreases were found in AST (2.0 and 4.0%), ALT (1.0, 2.0, and 4.0%), BUN (1.0 and 2.0%), and creatinine (1.0, 2.0, and 4.0%). Phosphorus (0.5 and 1.0%) and glucose levels (2.0%) were significantly increased.

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):

Significant decreases were seen in the organ weights and organ to body weight ratios for male rats in the high dose group (4.0%) for heart, liver, lung, spleen, and thymus when compared to the control group. The respective organ weights (treated)/organ weights (control) were: 1.03/1.27, 8.9/11.5, 1.46/1.86, 0.49/0.81, and 0.29/0.52 grams.
In the female rats only the thymus weight in the 4.0% dose group was significantly lower than control (0.32/0.42) grams.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The most significant histological changes were present in the kidney. Statistically significant increases in both incidence and severity of several kidney lesions occurred with an increasing dose of EG in the males (2.0 and 4.0% groups).
Significant histological renal changes included the following: tubular dilatation, characterized by widening of tubular lumens and variable flattening of lining epithelial cells; tubular degeneration, characterized by tubular epithelial cells with pyknotic or enlarged amphophilic nuclei, vacuolated and often slightly basophilic cytoplasm and intact cell membranes; tubular necrosis, characterized by loss of nuclei and disruption of cytoplasm and cell membranes; intratubular proteinaceous acellular eosinophilic material; acute inflammation; and calciumoxalate crystals. These crystals were pale yellow on hematoxylin and eosin stained sections and multicolored and birefringent under polarized light. There was increased incidence of intratubular crystals with increasing dose.
The only significant histological changes noted in the females were increased incidence of tubular dilatation and intra-tubular proteinaceous material.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

1 108 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

2 216 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Critical effects observed:

yes

Lowest effective dose / conc.:

2 216 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of this 10-day study, females demonstrated significantly decreased levels for hemoglobin, hematocrit, erythrocytes, and leukocytes in the 4% group. The most significant histopathological findings, seen predominantly in males, were kidney lesions which included calcium oxalate crystals in tubules and pelvic epithelium; tubular dilation and degeneration; intratubular proteinaceous material; and inflammation in tubules and pelvic epithelium. At the same dose of ethylene glycol, males had more kidney lesions and much higher incidence and severity of lesions than the females.

Reference 4

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The substances discussed in the analogue approach are ethylene carbonate and ethylene glycol, where ethylene carbonate (target substance) is rapidly metabolised to propylene ethylene (source substance) and carbon dioxide.
The hypothesis for the analogue approach is mainly based on the assumption that ethylene carbonate follows the documented metabolic pathway where cyclic organic carbonates are metabolized and converted to their respective glycols. The validity of the proposed read-across is further strengthened by the similarity in the toxicological profiles of both substances, indicating that ethylene carbonate as well as ethylene glycol do not exhibit systemic toxicity.
In order to support this evidence, an in vitro hydrolysis study was performed by Ehmer in 2015. In this study, propylene carbonate was incubated in Wistar rat blood over a time span of 30 minutes. The positive control item ethylene carbonate was also incubated in Wistar rat blood over a time span of 30 minutes. 35.5 % of the start concentration remained after 5 minutes of incubation. After 30 minutes 15.5% of the start concentration was observed. The hydrolysis product ethylene glycol was formed simultaneously from the reference item at concentrations that corresponded to its turnover/hydrolysis. The calculated half-life value for ethylene carbonate was 3.533 minutes. This corresponds to a turnover of 0.14 μmol/(ml x min). For both compounds the formation of the corresponding glycols was observed simultaneously.
Earlier data support the abovementioned study: in their work investigating the biotransformation of certain cyclic alkylene carbonates, Yang et al. (1998) identified a rat liver enzyme capable of hydrolyzing certain alkylene carbonates to CO2 and the respective alkylene glycol. In particular, Yang et al. conclude that “The mechanism previously outlined for the hydrolysis of imides appears to apply equally to the activity toward cyclic organic carbonates. The reaction would be expected to take the form of protonation of the carbonyl group of the carbonate, thereby providing a strong electrophilic center for the addition of water. Upon such addition, ring opening would be followed by elimination of CO2. The finding of this enzyme in rat liver provides a metabolic pathway for the conversion of cyclic organic carbonates to their respective glycols.” One of the substances used by Yang et al. to describe this phenomenon is ethylene carbonate. In support of the Yang et al. biotransformation data, a toxicokinetic study was conducted using ethylene carbonate. In addition, Hanley et al, (1989) also reported that the blood ethylene glycol levels in these rats were about 100-fold higher as compared to the parent compound ethylene carbonate. Whereas the half-life of ethylene glycol in the blood was approximately 2 hours, the half-life for ethylene carbonate was only 15 min, indicating rapid conversion of ethylene carbonate to ethylene glycol. Collectively, all these data strongly suggest that there is a similar metabolic pathway for the metabolism of ethylene and
propylene carbonate to their respective glycols.
Based on the weight of the evidence of the metabolism data indicating that alkylene carbonates are metabolised to the respective alkylene glycol, and the fact that ethylene carbonate is expected to be converted to ethylene glycol following absorption, it is proposed to use ethylene glycol as source substance to close data gaps of ethylene carbonate, target substance in an analogue approach. The detailed justification for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Details on results:

No mortality occurred in the 10-day study. Body weights were suppressed in a dose response fashion for males and females. Hemoglobin, hematocrit, erythrocytes, and leukocytes were all significantly decreased in female rats receiving 4% EG.
The most significant histopathological findings, seen predominantly in males, were kidney lesions which included calcium oxalate crystals in tubules and pelvic epithelium; tubular dilation and degeneration; intratubular
proteinaceous material; and inflammation in tubules and pelvic epithelium. At the same dose of ethylene glycol, males had more kidney lesions and much higher incidence and severity of lesions than the females.

Dose descriptor:

NOAEL

Effect level:

1 108 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

2 216 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Critical effects observed:

yes

Lowest effective dose / conc.:

2 216 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of this 10-day study, females demonstrated significantly decreased levels for hemoglobin, hematocrit, erythrocytes, and leukocytes in the 4% group. The most significant histopathological findings, seen predominantly in males, were kidney lesions which included calcium oxalate crystals in tubules and pelvic epithelium; tubular dilation and degeneration; intratubular proteinaceous material; and inflammation in tubules and pelvic epithelium. At the same dose of ethylene glycol, males had more kidney lesions and much higher incidence and severity of lesions than the females.

Reference 5

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

- Principle of test:
Ethylene glycol was administered in drinking water at concentrations of 0.5, 1.0, 2.0, and 4.0% for females and at 0.25, 0.5, 1.0, and 2.0% for males.
- Short description of test conditions: Animals were housed, two per cage, in an animal room maintained at 21-24°C and 40-60% relative humidity with 12 hour light-dark cycles. Rodent Chow N. 5001 (Ralston Purina Co., St. Louis, MO) and treatment solutions were available ad libitum.
- Parameters analysed / observed: At time of sacrifice necropsies were performed and tissues prepared for histological evaluation. Blood samples were taken for hematology and clinical chemistry determinations. Body weights were measured weekly. Water and food consumption were determined three times weekly.

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 2-852019
- ethylene glycol was administered via drinking water solutions which were prepared weekly by the addition of ethylene glycol to distilled water. The solutions were delivered in amber colored glass bottles with rubber stoppers and stainess steel sipper tubes with ball bearings to avoid loss of test material. The concentrations of solutions administered to the animals was confirmed and the stability of ethylene glycol in the drinking water was verified.

OTHER SPECIFICS: The chemical was analyzed for purity using gas chromatography and its composition confirmed by mass spectrometry. There were no impurities present within the detectable limits of the instrumentation.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Females (if applicable) nulliparous and non-pregnant: no data
- Age at study initiation: 85 days
- Weight at study initiation: average of 350 grams (males) and 245 grams (females)
- Fasting period before study: no data
- Housing: plastic cages with hardwood bedding (Ab-Sorb-Dri, Inc., Maywood, NJ), two animals per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week quarantine

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 °C
- Humidity (%): 40-60% relative humidity
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12hr/12hr

IN-LIFE DATES: not specified

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test item solutions were prepared weekly by the addition of ethylene glycol to distilled water. The solutions were delivered in amber colored glass bottles with rubber stoppers and stainless steel sipper tubes with ball bearing to avoid loss of test material.
Concentrations of solutions administered: 0, 0.5, 1.0, 2.0, 4.0% ethylene glycol solution (females); 0, 0.25, 0.5, 1.0, 2.0% ethylene glycol solution (males)

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): drinking water (distilled water)
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): distilled water was chosen as vehicle. The stability of ethylene glycol in the drinking water was verified.
- Concentration in vehicle: 0, 0.5, 1.0, 2.0, 4.0% ethylene glycol solution (females); 0, 0.25, 0.5, 1.0, 2.0% ethylene glycol solution (males)
- Amount of vehicle (if gavage): treatment solutions were available ad libitum

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

90 days

Frequency of treatment:

Treatment solution (drinking water) was available ad libitum during the exposure duration; Water bottles were changed on Monday, Wednesday and Friday of each week and consumption was measured at each change.

Dose / conc.:

0 other: % (v/v)

Remarks:

Males and Females

Dose / conc.:

0.25 other: % (v/v)

Remarks:

Males only
Theoretical concentration: 227 mg/kg/day

Dose / conc.:

0.5 other: % (v/v)

Remarks:

Males and Females
Theoretical concentration: 554 mg/kg/day

Dose / conc.:

1 other: % (v/v)

Remarks:

Males and Females
Theoretical concentration: 1108 mg/kg/day

Dose / conc.:

2 other: % (v/v)

Remarks:

Males and Females
Theoretical concentration: 2216 mg/kg/day

Dose / conc.:

4 other: % (v/v)

Remarks:

Females only
Theoretical concentration: 4432 mg/kg/day)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no data

Observations and examinations performed and frequency:

CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Rats were observed twice daily for clinical signs of changes in health

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed on the first day of dosing and weekly thereafter. Terminal body weights were measured at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: not applicable

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water bottles were changed on Monday, Wednesday and Friday of each week and consumption was measured at each change.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrifice (via cardiac puncture)
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: Hemoglobin, hematocrit, erythrocytes and leukocyte measurements were made with a Model ZBI Coulter Counter

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrifice (via cardiac puncture)
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: AST, ALT, LDH, cholesterol, phosphorus, calcium, glucose, BUN and creatinine

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Complete postmortem examinations were completed on all animals unless they were severely autolyzed.

HISTOPATHOLOGY: Yes
Tissues were fixed in 10% neutral buffered formalin and later trimmed, processed, embedded in paraffin, sectioned and stained with hematoxylin and eosin.
The following tissues were examined for histological changes from five animals in the control group and all animals in highest dose group having at least 60% of the animals surviving until terminal sacrifice: skin, mandibular and mesenteric lymph nodes, mammary glands, thigh muscle, sciatic nerve, femur and bone marrow, thymus, trachea, lungs, duodenum, jejunum, salivary gland, ileum, colon, cecum, rectum, liver, pancreas, spleen, kidneys, adrenals, urinary bladder, seminal vesicles, prostate, testes, epididymides, ovaries, uterus, nasal cavity and nasal turbinates, brain, pituitary, preputial and clitoral glands, Zymbal’s gland and any gross lesions. The target organ and any gross lesions were examined histologically from all remaining animals that survived until the terminal sacrifice. Lungs and any gross lesions were examined from all animals that died prior to the terminal sacrifice.

Other examinations:

Organ weights: (heart, kidney, liver, lung, spleen, brain, gonads and thymus) were measured at necropsy

Statistics:

Tukey's multiple comparison procedure was used for body weights, organ weights and organ/body weight ratio data. Kruskal-Wallis Rank Sum was used for the clinical chemistry and hematology data. Overall analyses were done by one factor analysis of variance. Values which differed from the vehicle control group at p<0.05 were considered significant. Statistical evaluation of the incidence of renal histopathological lesions was performed using Fisher's Exact Test. The significance of the severity of the lesions was determined by Pearson's Correlation Co-efficient and Correlation Analysis.

Clinical signs:

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

8/10 females and 2/10 males in the high dose group died prior to sacrifice.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Among animals surviving until sacrifice, only the high dose males showed a significant decrease in body weight.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not specified

Description (incidence and severity):

The actual consumption of EG by lower dose males and females in the 90-day study showed a tendency for reduced consumption while consumption levels for high dose groups were higher than anticipated with males in 2.0% group consuming 141% expected consumption and females in the 2.0 and 4.0% groups consuming 139% and 130%, respectively.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Leukocyte levels were significantly decreased in the females with counts of 3.4, 3.5 and 2.5 x 1E03/dL for the 0.5, 2.0 and 4.0% groups, respectively, compared to 5.0 x 1E03/dL in controls. No other significant results were found in males or females.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, significant increases were seen in LDH (1.0% group), phosphorus (2.0% group), BUN (2.0% group) and creatinine (1.0 and 2.0% group). Significant decreases were seen in males for ALT, calcium and glucose (1.0% group). In females, the only significant effects found were increased phosphorus (1.0% group) and glucose (0.5 and 4.0% groups).

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):

In the male rats there was a significant increase in the 1.0% and 2.0% group in kidney weights. A significant decrease was observed for heart, liver, and lung weights in male rats receiving 2.0% ethylene glycol. Organ weight/body weight ratios, in the 2.0% group for kidneys, brain and gonads were significantly increased, compared to controls. There were no significant changes observed in the females.

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The most significant histological changes were present in the kidney. Statistically significant increases in both incidence and severity of several kidney lesions occurred with an increasing dose of EG (2.0 and 4.0% groups in females; 1.0 and 2.0% groups in males). Males showed a greater number of lesions than females. Significant renal changes included: tubular dilatation; tubular degeneration; acute inflammation and birefringent crystals in tubules and pelvis epithelium. In contrast to the 10 day study, significant increases in intratubular proteinaceous material and tubular necrosis were not observed. Additional lesions which appeared with significantly increasing incidence and severity in the 90 day study were subacute inflammation; granulomatous inflammation characterized by giant cells within the interstitium; phagocytized crystals; dilatation of urinary pelvis; hyperplasia of kidney pelvis epithelium characterized by an increase in the number of layers of cells; and degeneration of pelvis epithelium.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOAEL

Effect level:

554 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

554 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Remarks on result:

other: A NOAEL value in females was not determinable as significant adverse effects in haematology have been observed in the lowest dose group of 0.5%

Critical effects observed:

yes

Lowest effective dose / conc.:

2 216 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

In the 90-day study 8/10 females and 2/10 males in the high dose group died prior to sacrifice. The most significant histological changes were present in the kidney. Males showed greater number of lesions than females. Significant renal changes included: tubular dilatation; tubular degeneration; acute inflammation and birefringent crystals in tubules and pelvis epithelium.

Reference 6

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

The substances discussed in the analogue approach are ethylene carbonate and ethylene glycol, where ethylene carbonate (target substance) is rapidly metabolised to ethylene glycol (source substance) and carbon dioxide. The validity of the proposed read-across is further strengthened by the similarity in the toxicological profiles of both substances. The detailed justification for the analogue approach is added to section 13 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Details on results:

In the 90-day study, 8/10 females and 2/10 males in the high dose group died prior to sacrifice.
The most significant histopathological findings, seen predominantly in males, were kidney lesions which included calcium oxalate crystals in tubules and pelvic epithelium; tubular dilation and degeneration; intratubular
proteinaceous material; and inflammation in tubules and pelvic epithelium. At the same dose of ethylene glycol, males had more kidney lesions and much higher incidence and severity of lesions than the females.

Dose descriptor:

NOAEL

Effect level:

554 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

554 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

haematology

Remarks on result:

other: A NOAEL value in females was not determinable as significant adverse effects in haematology have been observed in the lowest dose group of 0.5%

Critical effects observed:

yes

Lowest effective dose / conc.:

2 216 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Statistically significant increases in both incidence and severity of several kidney lesions were observed in the females in this study. However, this result is in contrast to results reported by other investigators. Ethylene Glycol toxicity needs further evaluation in terms of sex-dependent formation and fate of metabolites, possibly other than oxalate.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Lowest NOAEL observed in a chronic study

System:

urinary

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Even though the reference value is below the NOAEL identified in various studies in rats, ethylene glycol and hence also ethylene carbonate induce severe oxalate nephrosis; additionally, humans have been shown to be more susceptible following single exposure.

Additional information

Repeated dose toxicity: oral

No reliable data are available on ethylene carbonate. However, reliable read-across data from its metabolite ethylene glycol are used for the risk characterization of ethylene carbonate. The hypothesis of the analogue approach is mainly based on the assumption that ethylene carbonate follows the documented metabolic pathway were cyclic organic carbonates are metabolized and converted to their respective glycols. The validity of the proposed read-across is further strengthened by the similarity in the toxicological profile of both substances, indicating that ethylene carbonate as well as ethylene glycol do not exhibit systemic toxicity. In order to support this evidence, an in vitro hydrolysis study was performed by Ehmer (2015). The calculated half-life for ethylene carbonate was 3.533 minutes. This corresponds to a turnover of 0.14 µmol/(ml x min). Earlier data support the in vitro hydrolysis study (Yang et al., 1998 and Hanley et al., 1989).

Corley et al. (2008) exposed rats to 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. This study was used as critical study for DNEL derivation in the ethylene glycol dossier. Therefore, only this study has been taken over in the ethylene carbonate dossier. Additional studies on repeated dose toxicity via the oral route of ethylene glycol were available. Repeated oral exposure to sub-lethal doses of ethylene glycol may lead to oxalate nephrosis (renal destruction by Ca-oxalate crystals) and is therefore considered relevant for a STOT RE classification. In terms of renal toxicity, rats appeared to be more resistant than rabbits and less resistant than mice (NTP, 2004). However, also different strains of rats may show different sensitivities with more severity and more accumulation of oxalate in the kidneys of Wistar rats than Fischer rats (Cruzan et al., 2004). All subchronic and chronic oral studies in rats appear to converge in a NOAEL around 150 mg/kg bw and day (Corley et al., 2008).

In addition, Robinson et al. (1990) exposed male and female Sprague-Dawley rats to ethylene glycol in drinking water at concentrations of 0.5, 1.0, 2.0, and 4.0% for both sexes in the 10 -day study. Based on a projected consumption rate of 100 mL/kg/day, the respective doses on a mg/kg/day basis would be 554, 1108, 2216 and 4432. These dose levels were also used in the 90 -day study for females, but dose levels for the males in the 90 -day study were 0.25, 0.5, 1.0, and 2.0% (227, 554, 1108, and 2216 mg/kg/day). No mortality occurred in the 10 -day study. In the 90 -day study 8/10 females and 2/10 males in the high dose group died prior to sacrifice. Body weights were suppressed in a dose response fashion for males and females. Hemoglobin, hematocrit, erythrocytes, and leukocytes were all significantly decreased in female rats receiving 4% ethylene glycol for 10 days. The most significant histopathological findings, seen predominantly in males, were kidney lesions which included calcium oxalate crystals in tubules and pelvic epithelium; tubular dilation and degeneration; intratubular proteinaceous material; and inflammation in tubules and pelvic epithelium. At the same dose of ethylene glycol, males had more kidney lesions and much higher incidence and severity of lesions than the females.

Both the read-across study of Corley et al. (2008) and Robinson et al. (1990) are used to cover the endpoint requirements for repeated dose toxicity via the oral route in a weight of evidence approach.

Repeated dose toxicity: dermal and inhalation

No reliable data on repeated dose toxicity via the dermal and inhalation route are available for ethylene carbonate. However studies can be waived based on following justification: "Reliable chronic toxicity data (12 months exposure) are available from a study with ethylene glycol via the oral route. According to the REACH Regulation, a short-term and sub-chronic repeated dose toxicity test does not need to be conducted if a reliable chronic study is available, provided that an appropriate species and route of administration were used (Column 2 adaptation, Annexes VIII and IX respectively)".

Justification for classification or non-classification

According to the CLP regulation, substances can be classified as Category 2 for STOT RE if toxic effects with relevance to human health have been observed. Hence, based on responsible care, ethylene glycol and ethylene carbonate are classified as STOT RE category 2, H373 according to the criteria of the CLP Regulation.