Ethylene carbonate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction:
No reliable data are available with ethylene carbonate. However, a parental and offspring NOAEL of greater than 1000 mg/kg bw was observed in a three-generation study with male and female Fischer 344 rats for ethylene glycol, the predominant metabolite of ethylene carbonate (DePass et al., 1986).

In addition, Gulati et al. (1984) reported a fertility study and found a NOEL of 1000 mg/kg for parental and F1 male and female mice. The authors deemed ethylene glycol a "weak reproductive toxicant, but a potential teratogen".

Link to relevant study records

Reference

Reference 1

Endpoint:

three-generation reproductive toxicity

Remarks:

based on test type

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

Reason / purpose for cross-reference:

read-across: supporting information

Diet consumption and body weight: Throughout the study there was no effect of the analogue (ethylene glycol) treatment on body weight gain or diet consumption, nor was there any mortality among parental rats. The calculated dosages based on the nominal concentrations of the analogue substance in the diet were generally very close to the dosage goals. The weekly calculated dosages ranged from 1.0 to 1.3, 0.2 to 0.3, and 0.04 to 0.05 g/kg/day for males and from 0.9 to 1.2, 0.2 to 0.3, and 0.04 to 0.06 g/kg/day for females.
Reproductive indices: No treatment-related effect was observed for any of the indices. Also, the treatment with the analogue substance did not affect neonatal body weight at days 4, 14, or 21 post partum.

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: There were no reproductive effects associated with the inclusion of as much as 1.0 g/kg/day of the analogue in the diet

Histopathologic findings: There were no treatment-related histopathologic findings in F2 parents or in F3 weanlings. Although the kidney has been shown to be the primary target organ for EG-induced toxicity, there was no increase in the incidence or severity of kidney lesions in this study. One high dose F2 animal of each sex had mild focal interstitial nephritis. However, this condition was also seen in a control male and a control female. Unilateral hydronephrosis occurred in another high-dose F2 male. In addition, mild focal tubular hyperplasia was observed in one high-dose male F3 pup but was also diagnosed in two control male pups.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: There were no reproductive effects associated with the inclusion of as much as 1.0 g/kg/day of the analogue in the diet

Reproductive effects observed:

not specified

Reference 2

Endpoint:

fertility, other

Remarks:

based on test type

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

Reason / purpose for cross-reference:

read-across: supporting information

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effects on litter size, pup weight or sex ratio

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: no effects on litter size, pup weight or sex ratio

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction:

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).

DePass et al. (1986) reported a three-generation reproduction study. Male and female rats were given orally (feed) daily doses of 40, 200 and 1000 mg ethylene glycol/kg/day. No reproductive effects associated with the inclusion of as much as 1000 mg/kg/day of EG in the diet were found. The NOAEL for parental animals and for offsprings was found to be greater than 1000 mg/kg.

Gulati et al (1984) reported a fertility study and found a NOEL of 1000 mg/kg for parental and F1 male and female mice. Ethylene glycol was administered in drinking water in concentrations of approximately 500, 1000 and 2000 mg/kg/day. Exposure to ethylene glycol resulted in a small but significant decrease in the number of litters per breeding pair, in the number of live pups per pair and in the live pup weight. A significant number of pups in the 1.0% dose group (2000 mg/kg) were born with distinct facial deformities. In the retained litters at this dose, the facial deformities were more obvious with age. These malformed animals also exhibited fused ribs and shortened nasal, parietal, and/or frontal bones of the skull. When pups from the high dose group were raised to adulthood (with continued exposure to ethylene glycol) and mated, they exhibited decreased mating and fertility indices relative to controls handled in the same manner, but there were no effects on litter size, pup weight or sex ratio. The authors deemed ethylene glycol a "weak reproductive toxicant, but a potential teratogen".

Effects on developmental toxicity

Description of key information

Ethylene carbonate (EC) was administered orally by gavage to SD rats at doses of 750, 1500 and 3000 mg/kg/day on Days 6 through 15 of gestation (equivalent to OECD guideline 414; Pharmakon Research International, 1991). Cesarean section was performed on each dam post copulation Day 20. Statistically significant decreases were observed in the group mean fetal body weights in the 1500 and 3000 mg/kg dose groups. These decreases were considered biologically significant and related to the administration of the test substance. Moreover, thirty three fetuses with malformations were detected during the study. Malformations were observed in one control fetus (0.3 %) from one litter (3.8 %), one low dose fetus (0.3 %) from one litter (3.8 %) and thirty one high dose fetuses (9.2 %) from eleven litters (45.8 %). A statistically significant increase was observed in the total number of fetal malformation and in the number of litters with malformations in the 3000 mg/kg dose group. The preponderance of skeletal variations were observed in the vertebrae and sternebrae. A statistically significant increase in the number of fetuses exhibiting incomplete ossification of the 1^st, 2^nd, 3^rdor 4^thsternebrae and unossification of the 6^thsternebrae was noted in the 1500 and 3000 mg/kg dose groups. In addition, a significant number of fetuses exhibiting bipartite thoracic vertebra was noted in the 3000 mg/kg dose group. Although not statistically analyzed (no variance in the control group), an increase in fetuses exhibiting bipartite lumbar vertebra was also noted in the 3000 mg/kg dose group.

Concerning the dams, there were no clinical signs observed in the vehicle control group. One dam in the 750 mg/kg dose group exhibited post dose salivation on Day 15 of gestation. In the 1500 mg/kg dose group, one dam displayed post dose salivation on Day 12, one displayed dyspnea and subsequent death on Day 6, and one exhibited chromodacryorrhea on Days 11 through 20. Thirteen of the dams in the 3000 mg/kg dose group exhibited post dose salivation during various intervals of the study. In addition, one of these females exhibited alopecia and another displayed decreased activity, decreased body tone, dyspnea and subsequent death.

Two dams died during the study. One dam from the 1500 mg/kg dose group died on Day 6 of gestation, and necropsy revealed inflated lungs. The other dam, from the 3000 mg/kg dose group, was found dead on Day 11 of gestation. Necropsy of this animal revealed discolored lungs and test substance visible in stomach. Both deaths were attributed to technical error and not the result of test substance toxicity.

One hundred and eight rats were mated and placed in study. 26/27 (96.3 %) were gravid in the vehicle control and 750 mg/kg dose groups, 27/27 (100 %) were gravid in the 1500 mg/kg dose group and 25/27 (92.6 %) were gravid in the 3000 mg/kg dose group. Statistically significant decreases were observed in the group mean dam body weight changes for the 3000 mg/kg dose group during Days 6 through 9, 6 through 15 and 0 through 20 of gestation. These decreases were considered biologically significant. In addition, a significant decrease was observed in the group mean dam body weight changes for the 750 mg/kg dose group during Days 6 through 9 of gestation. This decrease was observed only during one time point and was considered coincidental and not biologically significant.

Based on the results of the study, the NOEL for maternal toxicity was 1500 mg/kg/day and the NOEL for developmental toxicity was 750 mg/kg/day (Pharmakon Research International, 1991).

 

No further reproductive or developmental toxicity data are available on ethylene carbonate. However, reliable read-across data from its metabolite ethylene glycol (EG) are used for the risk characterization of EC at the endpoint developmental toxicity. The hypothesis of the analogue approach is mainly based on the assumption that EC 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 EC as well as EG 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 EC 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).

 

Ethylene glycol (EG) has been evaluated for effects upon pre-natal development following exposure during the period of organogenesis in both rodent (mouse, rat) and non-rodent (rabbit) species.

EG causes developmental toxicity - characterised by craniofacial and axial-skeletal malformations and variations - in mice (Tyl, 1989; dosing up to 1500 mg/kg daily) and rats when administered by oral gavage during the period of organogenesis. In contrast, the administration of EG to pregnant rabbits during organogenesis at doses up to 2000 mg/kg bw/d by oral gavage had no effect upon the progeny, although the highest dose was associated with substantial (42%) maternal mortality.

Subsequent investigations, both in vivo and in vitro, have established that the developmental toxicity of EG in rats is related to the accumulation of glycolic acid (GA) in the blood and metabolic acidosis. The toxicity of GA, both in vivo and in vitro, is exacerbated under acidic conditions and is related to fetal disposition. When EG was administered to rats and rabbits at a developmentally toxic dose (1000 mg/kg bw/d, p.o.), GA was found to be preferentially distributed into the rat embryo compared to the maternal blood (embryo:blood concentration 1.54) whereas this was not the case in the rabbit (embryo:blood concentration 0.31).

Recent investigation demonstrated that GA uptake into the rat embryo occurs predominantly by a specific, pH-dependent, active uptake transporter protein, consistent with the proton-linked monocarboxylate transporters (MCT).

Given that GA disposition between the maternal blood and the embryo is driven by the polarity of MCT1 and MCT4 isoforms in the placenta, that GA is sequestered in the rat and not the rabbit, and that the rabbit and human placenta show similar polarity to each other and opposite to that of the rodent, it is proposed that the rabbit is the most appropriate species for the basis of classification. As such, and since EG is not a developmental toxicant in the rabbit, no classification is warranted. Since read-across is considered valid at this endpoint, the conclusion of EG holds also true for EC.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 7, 1990 to May 17, 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Similar to OECD 414, but animals were treated from Days 6-15 of gestation rather than administered daily from implantation (e.g., day 5 post mating) to the day prior to scheduled caesarean section.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

animals were treated from Days 6-15 of gestation rather than administered daily from implantation (e.g., day 5 post mating) to the day prior to scheduled caesarean section.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Wilmington, Massachusetts
- Age at study initiation: 109 days
- Weight at study initiation: 232-293 grams
- Housing: Animals were housed individually in stainless steel 1/2 inch wire mesh cages sized in accordance with the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Animal Resources, National research Council. Nesting materials were not provided as sacrifice was scheduled prior to anticipated parturition.
- Diet (e.g. ad libitum): Purina Certified Rodent Chow was available ad libitum.
- Water (e.g. ad libitum): Fresh tap water was available ad libitum.
- Acclimation period: 11 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): Every attempt was made to maintain a relative humidity of 30-70 %. Humidity reading below 30 % occurred on October 27, 1990 (26 %) and on October 29, 1990 (28 %). These readings did not adversely affect the animals in the study.
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 1990-11-07 To: 1990-11-30

Route of administration:

oral: gavage

Vehicle:

other: deionized water

Details on exposure:

VEHICLE
- Amount of vehicle (if gavage): 5 ml/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the test substance vehicle mixtures and vehicle control were collected for submission to the sponsor on November 13, 1990 (Day 6) and November 22, 1990 (Day 15) to determine the concentration of the test substance in the vehicle mixture. Stability samples of the test substance were sampled for submission on November 13, 1990 and December 3, 1990.

Details on mating procedure:

-Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: one male to one or two females
- Length of cohabitation: Animals were cohoused until the females exhibited evidence of copulation.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Animals were treated by oral gavage on Days 6-15 of gestation.

Frequency of treatment:

Once daily

Duration of test:

Cesarean section was performed on each dam post copulation, Day 20.

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Dose / conc.:

3 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

27

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in the assay were based upon the results of two Dose-Range finding studies and were selected by the sponsor. The range-finding experiments were as follows:
1. Thirty six female Sprague Dawley rats were mated. Five groups of six rats were administered the test substance at doses of 125, 250, 500, 1000 and 2000 mg/kg/day, orally, by gavage on Days 5-15 of gestation. An addition group of six rats received deionized water at a constant volume of 5 ml/kg/day and served as a vehicle control. Cesarean section was performed on each dam post copulation Day 20. The uterus of each female was excised and weighed and the number of corpora lutea, viable and non viable fetuses, early resorptions, late resorptions and total implantations were recorded. The fetuses were examined for gross external morphological observations.

Based upon the findings of the first range-finding study, a second dose range finding study was performed, as described below.
2. Twenty four female Sprague Dawley rats were mated. Three groups of six rats were administered the test substance at doses of 3000, 4000 and 5000 mg/kg/day, orally, by gavage on Day 6-15 of gestation. An additional group of six rats received deionized water at a constant volume of 5 ml/kg/day and served a vehicle control. Same examinations were performed as described above for the first range finding experiment.

Based upon the results of the second range finding study, the main study was conducted at dose levels of 0, 750, 1500 and 3000 mg/kg/day.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: Pertinent changes and signs of toxicity including mortality were recorded.


BODY WEIGHT: Yes
- Time schedule for examinations: The females were weighed the day copulation was confirmed (Day 0) and on Days 6, 9, 12, 15 and 20 of presumed gestation or the day found dead. On Day 20 both actual and corrected body weights (subtract weight of gravid uterus) were recorded.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal was recorded for gestation days 0-6, 6-9, 9-12, 12-15, and 15-20. Individual and mean food consumption was expressed in grams.
- The amount of test substance administered to the individual females during the dosing period was based on the most recent individual body weights.



POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: A necropsy was performed and the uterus was examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

Statistical analyses compared the treatment groups to the control group with the level of significance at p

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed in the vehicle control group. One dam in the 750 mg/kg dose group exhibited post dose salivation on Day 15 of gestation. In the 1500 mg/kg dose group, one dam displayed post dose salivation on Day 12, one displayed dyspnea and subsequent death on Day 6, and one exhibited chromodacryorrhea on Days 11 through 20. Thirteen of the dams in the 3000 mg/kg dose group exhibited post dose salivation during various intervals of the study. In addition, one of these females exhibited alopecia and another displayed decreased activity, decreased body tone, dyspnea and subsequent death.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two dams died during the study. One dam from the 1500 mg/kg dose group died on Day 6 of gestation. The other dam was from the 3000 mg/kg dose group and was found dead on Day 11 of gestation. Both deaths were attributed to technical error and not the result of test substance toxicity.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases were observed in the group mean dam body weight changes for the 3000 mg/kg dose group during Days 6 through 9, 6 through 15 and 0 through 20 of gestation. These decreases were considered biologically significant. In addition, a significant decrease was observed in the group mean dam body weight changes for the 750 mg/kg dose group during Days 6 through 9 of gestation. This decrease was observed only during one time point and was considered coincidental and not biologically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in the amount of food consumed among the four dose groups.

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:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One dam from the 1500 mg/kg dose group died on Day 6 of gestation, and necropsy revealed inflated lungs. The other dam was from the 3000 mg/kg dose group and was found dead on Day 11 of gestation. Necropsy of this animal revealed discolored lungs and test substance visible in stomach.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Description (incidence and severity):

None of the dams aborted

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed in the mean number of implantations or the number and percentage of pre- or post-implantation losses.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

No statistically significant differences in mean number of late and early resorptions

Dead fetuses:

no effects observed

Description (incidence and severity):

No statistically significant differences in the mean number of viable fetuses

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

None of the dams delivered early

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

One hundred and eight rats were mated and placed in study. 26/27 (96.3 %) were gravid in the vehicle control and 750 mg/kg dose groups, 27/27 (100 %) were gravid in the 1500 mg/kg dose group and 25/27 (92.6 %) were gravid in the 3000 mg/kg dose group. 

Other effects:

not specified

Details on maternal toxic effects:

There were no clinical signs observed in the vehicle control group. One dam in the 750 mg/kg dose group exhibited post dose salivation on Day 15 of gestation. In the 1500 mg/kg dose group, one dam displayed post dose salivation on Day 12, one displayed dyspnea and subsequent death on Day 6, and one exhibited chromodacryorrhea on Days 11 through 20. Thirteen of the dams in the 3000 mg/kg dose group exhibited post dose salivation during various intervals of the study. In addition, one of these females exhibited alopecia and another displayed decreased activity, decreased body tone, dyspnea and subsequent death.
Two dams died during the study. One dam from the 1500 mg/kg dose group died on Day 6 of gestation, and necropsy revealed inflated lungs. The other dam was from the 3000 mg/kg dose group and was found dead on Day 11 of gestation. Necropsy of this animal revealed discolored lungs and test substance visible in stomach. Both deaths were attributed to technical error and not the result of test substance toxicity.
One hundred and eight rats were mated and placed in study. 26/27 (96.3 %) were gravid in the vehicle control and 750 mg/kg dose groups, 27/27 (100 %) were gravid in the 1500 mg/kg dose group and 25/27 (92.6 %) were gravid in the 3000 mg/kg dose group.
Statistically significant decreases were observed in the group mean dam body weight changes for the 3000 mg/kg dose group during Days 6 through 9, 6 through 15 and 0 through 20 of gestation. These decreases were considered biologically significant. In addition, a significant decrease was observed in the group mean dam body weight changes for the 750 mg/kg dose group during Days 6 through 9 of gestation. This decrease was observed only during one time point and was considered coincidental and not biologically significant.

Dose descriptor:

NOEL

Effect level:

ca. 1 500 mg/kg bw/day (actual dose received)

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases were observed in the group mean fetal body weights in the 1500 and 3000 mg/kg dose groups. These decreases were considered biologically significant and related to the administration of the test substance.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no fetal deaths recorded at cesarean section.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No statistically significant differences in fetal sex distribution

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decreases were observed in the group mean fetal body weights in the 1500 and 3000 mg/kg dose groups. These decreases were considered biologically significant and related to the administration of the test substance.

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Thirty three fetuses with malformations were detected during the study. Malformations were observed in one control fetus (0.3 %) from one litter (3.8 %), one low dose fetus (0.3 %) from one litter (3.8 %) and thirty one high dose fetuses (9.2 %) from eleven litters (45.8 %).

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase was observed in the total number of fetal malformation and in the number of litters with malformations in the 3000 mg/kg dose group. The preponderance of skeletal variations were observed in the vertebrae and sternebrae. A statistically significant increase in the number of fetuses exhibiting incomplete ossification of the 1st, 2nd, 3rd or 4th sternebrae and unossification of the 6th sternebrae was noted in the 1500 and 3000 mg/kg dose groups. In addition, a significant number of fetuses exhibiting bipartite thoracic vertebra was noted in the 3000 mg/kg dose group. Although not statistically analyzed (no variance in the control group), and increase in fetuses exhibiting bipartite lumbar vertebra was also noted in the 3000 mg/kg dose group.

Visceral malformations:

not specified

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Statistically significant decreases were observed in the group mean fetal body weights in the 1500 and 3000 mg/kg dose groups. These decreases were considered biologically significant and related to the administration of the test substance.
Moreover, thirty three fetuses with malformations were detected during the study. Malformations were observed in one control fetus (0.3 %) from one litter (3.8 %), one low dose fetus (0.3 %) from one litter (3.8 %) and thirty one high dose fetuses (9.2 %) from eleven litters (45.8 %).
A statistically significant increase was observed in the total number of fetal malformation and in the number of litters with malformations in the 3000 mg/kg dose group. The preponderance of skeletal variations were observed in the vertebrae and sternebrae. A statistically significant increase in the number of fetuses exhibiting incomplete ossification of the 1st, 2nd, 3rd or 4th sternebrae and unossification of the 6th sternebrae was noted in the 1500 and 3000 mg/kg dose groups. In addition, a significant number of fetuses exhibiting bipartite thoracic vertebra was noted in the 3000 mg/kg dose group. Although not statistically analyzed (no variance in the control group), and increase in fetuses exhibiting bipartite lumbar vertebra was also noted in the 3000 mg/kg dose group.

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Sex:

not specified

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Results of first range-finding study: There were no pharmacotoxic signs observed in the vehicle or 125 mg/kg/day dose groups. Post dose salivation was observed from one female each in the 250 and 500 mg/kg/day dose groups, three females from the 1000 mg/kg/day group and five from the 2000 mg/kg/day dose group. None of the females died during the study. There were no statistically significant differences observed in the group mean dam body weights, body weight gains or food consumption data that was attributable to the administration of the test substance. There were no statistically significant differences observed in the mean number of implantations, corpora lutea, viable fetuses, late resorptions, numbers of the pre and post implantation losses or the percentages of theses losses. No gross external malformations were detected during the study.

Results of second range-finding study: There were no pharmacotoxic signs observed in the vehicle control group. Maternal toxicity was observed in all dose groups as evidenced by suppressed body weight, body weight gain and reduced food consumption. Several clinical signs were observed in the 3000 and 5000 mg/kg/day dose groups. These signs included dyspnea, abnormal gait, abnormal stance, post dose salivation, decreased activity, ptosis, piloerection and prostration. Two dams died during the study. These deaths were a result of technical error and not the administration of the test substance. There were no statistically significant differences observed in the mean number of implantations, corpora lute, viable fetuses, late resorptions, numbers of pre and post implantation losses or the percentages of these losses. Twenty nine malformed fetuses were detected during the study. These were detected in 2 of 68 low dose fetuses from 1 of 5 litters, 18 of 89 mid dose fetuses from 2 of 6 litters, and 9 of 50 high dose fetuses from 3 of 4 litters.

Conclusions:

One hundred and eight female Sprague Dawley rats were mated for use in the study to determine the potential hazard of the test substance to the unborn which may arise from exposure to the mother during pregnancy. The test substance was administered orally by gavage, at doses of 750, 1500 and 3000 mg/kg/day on Days 6 through 15 of gestation. Cesarean section was performed on each dam post copulation, Day 20. Based on the results of the study, the no observed effect level for maternal toxicity was 1500 mg/kg/day and the no observed effect level for developmental toxicity was 750 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

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

Reason / purpose for cross-reference:

read-across: supporting information

Details on maternal toxic effects:

Details on maternal toxic effects:
Maternal toxicity was observed at 2000 mg/kg/day, expressed as 42.1% mortality (8 of 17 pregnant animals), as well as an increase in early deliveries with three at this dose (and one each at all other doses). One doe at 2000 mg/kg/day aborted on GD 20 (no litters were aborted at any other doses). lncreases in early deliveries and abortion are usually considered indicative of maternal stress in rabbits. Pregnancy rate was high and equivalent across all groups from 0 - 1000 mg/kg/day (95.5, 95.7, 91.3, and 95.2%, respectively); pregnancy rate was slightly lower (8 1.8%) at 2000 mg/kg/day. Periodic maternal body weights and weight changes were statistically equivalent across all groups for all intervals evaluated. Maternal water consumption, expressed as g/animal/day or as g/kg/day was also statistically equivalent across all groups for all intervals, although water consumption appeared slightly increased for all EG-dosed groups during the treatment period but not in a manner related to dose. No clear treatment-related clinical signs of toxicity were observed. The 8 does which died at 2000 mg/kg/day died on GD 9 (one doe) and 11 (two does) and on GD 13, 14, 19, 21 and 25 (one doe each). At scheduled necropsy on GD 30 there were no significant effects of treatment on corrected (for uterine weight) maternal gestational weight change, gravid uterine weight, liver weight, or kidney weight at any dose. However, maternal absolute kidney weight (but not relative weight) was slightly increased at 2000 mg/kg/day to 106.3% of the control value for the right kidney (left kidney value was 107.6% of control). Histologic evaluation of maternal kidneys revealed treatment-related renal lesions only at 2000 mg/kg/day. The lesions were limited to the cortical renal tubules and included intraluminal crystals (appearance consistent with oxalate), epithelial necrosis, and tubular dilatation and degeneration. The most severe findings, crystals (designated "marked") and necrosis, were observed in the does which died on study, but the renal tubular necrosis observed in these animals was not a postmortem event. The cause of death in these animals was determined to be renal failure. There were no treatment-related effects on any gestational parameters, including no effects on number of ovarian corpora lutea, on total non-live or live implantation sites per litter, or on pre- or post-implantation loss. There was a statistically significant increase in the number of corpora lutea at 500 mg/kg/day associated, as expected, with a slight increase in the number of implantation sites/litter and a slight increase in live litter size. This finding was not observed at higher doses and is considered most likely due to biologic variation. The values at 500 mg/kg/day are still well within historical control values for these parameters.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
There were no statistically significant or biologically relevant differences among groups for prenatal mortality, expressed as resorptions or dead fetuses, or for prenatal toxicity, expressed as fetal body weight/litter for all fetuses or for male and female fetuses separately. There were no treatment-related alterations in incidence of malformations pooled as external, visceral (including craniofacial), or skeletal, or as total malformations or variations. Examination of fetal malformations and variations by individual findings also indicated no findings which were treatment- or dose-related and none which appeared predominantly or exclusively at higher doses.

Dose descriptor:

NOAEL

Effect level:

2 000 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 3

Endpoint:

developmental toxicity

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

Reason / purpose for cross-reference:

read-across: supporting information

Dose descriptor:

NOEL

Effect level:

1 500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no maternal toxicity at 500 and 1500 mg/kg/day.

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Reference 4

Endpoint:

developmental toxicity

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

Reason / purpose for cross-reference:

data waiving: supporting information

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

500 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Dose descriptor:

LOAEL

Remarks:

fetotoxicity

Effect level:

1 000 mg/kg bw/day

Based on:

test mat. (total fraction)

Sex:

not specified

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEL

Remarks:

teratogenicity

Effect level:

2 500 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

The lowest NOAEL for developmental toxicity has been observed in mice. The NOAEL for rabbits, after exposure to ethylene glycol, is above 2000 mg/kg/d and 750 mg/kg bw in rats after exposure to ethylene carbonate.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Ethylene carbonate (EC) was administered orally by gavage to SD rats at doses of 750, 1500 and 3000 mg/kg/day on Days 6 through 15 of gestation (equivalent to OECD guideline 414; Pharmakon Research International, 1991). Cesarean section was performed on each dam post copulation Day 20. Based on the results of the study, the NOEL for maternal toxicity was 1500 mg/kg/d and the NOEL for developmental toxicity was 750 mg/kg/d (Pharmakon Research International, 1991).

 

The read-across substance ethylene glycol (EG) has been evaluated for effects upon pre-natal development following exposure during the period of organogenesis in both rodent (mouse, rat) and non-rodent (rabbit) species. EG caused developmental toxicity at high doses after gavage administration in mice and rats, but not after administration via dermal route, in the diet or in drinking water. Developmental effects were seen in two studies in mice with nose only and whole body inhalation exposure, but only at EG aerosol concentrations that would be intolerably high for humans. No effects on development were observable in rabbits.

 

Mouse

Administration of EG (0, 750, 1500, or 3000 mg/kg bw/d; p.o.) to pregnant CD-1 mice caused increased incidences of skeletal malformations at all doses, and increased incidence of external and visceral malformations at the high-dose level. As with the rat, external malformations were predominantly neural tube defects (meningoencephalocele; exencephaly) and facial malformations (cleft lip and palate; facial cleft), while skeletal malformations involved the ribs, vertebral arches, and centra. Fetal body weight was reduced at all doses, but live litter size was only reduced at the high-dose level (Price et al., 1985). These findings were also subject to a follow-up study to define the NOAEL for effects upon development. Pregnant CD-1 mice were administered EG (0, 50, 150, 500, or 1500 mg/kg bw/d; p.o. GD 6- 15) resulting in skeletal malformations and variations and reduced fetal weight at the highest dose, and an increase in the incidence of one skeletal variation at 500 mg/kg/day. The authors identified a NOAEL of 150 mg/kg/d for developmental toxicity and a NOEL of 1500 mg/kg bw/d for maternal toxicity (Neeper-Bradley et al., 1995).

 

In a FACB study in which CD-1 mice were continuously administered 1% EG in the drinking water (equivalent to approx. 1640 mg/kg bw/d) and repeatedly bred, adult offspring of EG-treated dams had unusual facial features, characterized by shortened snout and wide-set eyes. Skeletal examination revealed defects of the skull, sternebrae, ribs, and vertebrae. Gross malformations, such as cleft lip, were also noted in neonates from both the F0 and F1 generations. No effects were noted in animals that received 0.25% or 0.5% EG (equivalent to approx. 410 or 840 mg/kg bw/d) in the drinking water (NTP, 1984).

 

Whole body exposure of CD-1 mice to EG aerosol (0, 150, 1000, 2500 mg/m³ for 6 h/day) was embryotoxic, eliciting malformations and reduced fetal body weight at the mid- and high-exposure concentrations (Tyl et al., 1995a). The potential for oral exposure to EG by grooming of the fur was investigated by exposing CD-1 mice to EG aerosol either nose-only (0, 500, 1000, 2500 mg/m³, 6 h/day) or whole body (0, 2100 mg/m³, 6 h/day). Fetal body weight was reduced at the highest concentration by both exposure regimens, although the majority of skeletal malformations were only caused by whole body exposure (Tyl et al., 1995b). By comparison, the maximum tolerable inhalation regimen in human tests was 308 mg/m³ aerosol (2 breaths) (Wills et al., 1974). Furthermore, dermal exposure of CD-1 mice to EG (0, 404, 1677, or 3549 mg/kg bw/d, the latter equivalent to 100% undiluted EG) was without effect on the progeny (Tyl et al., 1995c). 

 

Rat

Administration of EG (0, 1250, 2500, or 5000 mg/kg bw/d; p.o.) to pregnant CD rats increased the incidences of visceral malformations at all doses, increased the incidences of skeletal malformations at the mid- and high-dose levels, and increased the incidence of external malformations at the high-dose level. External malformations were predominantly neural tube defects (meningoencephalocele; exencephaly; meningocele) and facial malformations (cleft lip and palate; anophthalmia; micrognathia), while skeletal malformations involved the ribs, arches, and centra, and visceral anomalies were predominantly of the great vessels. Fetal body weight and live litter size were also reduced at the mid- and high-dose levels (Price et al., 1985). In a subsequent study to define the NOAEL for effects upon development, pregnant CD rats were administered EG (0, 150, 500, 1000, or 2500 mg/kg bw/d; p.o.). This resulted in external and skeletal malformations at the highest dose. Skeletal malformations and variations (reduced ossification) were noted at 1000 mg/kg bw/d, while the only statistically significant finding at 500 mg/kg bw/d was poor ossification of the supraoccipital bone. Regarding maternal toxicity, NOEL was considered to be 1000 mg/kg bw/d. However, regarding developmental toxicity NOAEL was considered to be 500 mg/kg bw/d (Neeper-Bradley et al., 1995).

 

Maronpot et al (1983) administered EG in the diet to F344 rats at doses of up to 1000 mg/kg bw/d. This was without effect upon the progeny, indicating that the effects of EG are attenuated by non-bolus dosing regimens. This conclusion is supported by the results of a study in which the same overall dose of EG (0, 1000, 2000 mg/kg bw/d; s.c.) was administered as a bolus dose or by infusion. Bolus dosing resulted in skeletal malformations and variations at both dose levels, whereas infusion did not (Carney et al., 2011).

 

Tyl et al. (1995a) reported a teratogenic study with rats and mice. Rats were given whole body inhalative (aerosol) concentrations of 150, 1000 or 2500 mg/m³ for 6 hours per day from gestation day 6 - 15. The NOAEC for maternal toxicity was found to be 1000 mg/m³ air 6 h/d and the NOAEC for developmental toxicity was found to be 150 mg/m³ air 6 h/d. Exposure of rats to EG glycol aerosol during organogenesis resulted in minimal maternal toxicity in rats at 2500 mg/m³ 6 h/d and minimal fetotoxicity in at 1000 and 2500 mg/m³ 6 h/d. There was no maternal or embryofetal toxicity at 150 mg/m³ 6 h/d (NOEL) and no teratogenicity at any aerosol concentration employed.

 

NTP (1988) reported a developmental toxicity in rats which were gavaged with daily doses of 250, 1250 or 2250 mg/kg bw/d from gestation day 6 - 20. Administration of EG to timed-mated rats resulted in no observed toxicity at 250 mg/kg bw/d. At 1250 mg/kg bw/d, the gestational period was lengthened, and maternal renal pathology was noted, but no adverse developmental effects were observed. At 2250 mg/kg bw/d, reduction of maternal body weight, weight gain, kidney weight and postpartum uterine weight were observed. In addition, gestation was lengthened and maternal renal pathology was present. Developmental toxicity at 2250 mg/kg bw/d included reduced pup body weight, reduced viability and increased malformation incidence (primarily hydrocephaly and abnormalities of the axial skeleton), but adverse effects upon other indices of postnatal development were not observed. The NOAEL was found to be 250 mg/kg bw/d for maternal and developmental toxicity.

 

Rabbit

Administration of EG to New Zealand White rabbits (0, 100, 500, 1000, or 2000 mg/kg bw/d; p.o.) caused mortality in 42% of the does as a result of renal failure. In the offspring, no effects upon survival, fetal weight, or the incidences of external, visceral, or skeletal malformations or variations were observed at any dose level, including the progeny of surviving does treated with 2000 mg EG/kg/day. Altogether, EG resulted in profound maternal toxicity at 2000 mg/kg/day (42% mortality; three early deliveries and one spontaneous abortion) associated with renal pathology (Tyl et al., 1993).

 

The Role of metabolism in the aetiology of EG developmental toxicity

Following absorption, EG is rapidly metabolised by oxidative pathways (Corley et al., 2005). At low doses, metabolism is extensive, and the major elimination products are exhaled carbon dioxide and urinary parent glycol and metabolites. However, the oxidation of glycolic acid (GA) to glyoxylic acid becomes saturated at doses in the range 125–500 mg/kg bw, resulting in accumulation of GA in the blood and increased excretion in the urine, with no significant difference between pregnant and non-pregnant female CD rats (Corley et al., 2005; Pottenger et al., 2001). The accumulation of GA in the blood is associated with metabolic acidosis. EG is of low inherent toxicity to the rat embryo. The NOAEC for morphological changes in rat embryos exposed to EG in vitro is in excess of 50 mM (Carney et al., 1996; Grafton & Hansen, 1987; Klug et al, 2001), this is in comparison to the maximum blood concentration of 14 mM EG following an oral bolus dose of 1000 mg/kg bw (Pottenger et al., 2001). Klug et al. (2001) evaluated the toxicity of EG metabolites as well as the parent glycol in rat whole embryo culture (WEC), determining that all metabolites were more potent in inducing anomalies and growth retardation. Comparison of the NOAEC/LOAEC with measured levels of metabolites in blood following administration of EG to female rats (Frantz et al., 1996a, 1996b; Pottenger et al., 2001) indicates that GA is the proximate toxicant (Carney et al., 1996; Klug et al., 2001). In two separate studies the NOAEC and LOAEC for GA were identified as 2.5 mM and 12.5 mM, respectively by Carney et al. (1996) and as 1 mM and 3 mM by Klug et al. (2001). In comparison, the maximum blood glycolate concentration following a dose of 500 or 1000 mg/kg bw EG to the pregnant CD rat is 1.7 mM or 4.8 mM, respectively (Pottenger et al., 2001).

When administered to pregnant CD rats, GA induced a spectrum of skeletal malformations similar to that of EG (Carney et al., 1999; Munley et al., 1999). To distinguish the roles of glycolate anion and acidosis in the aetiology of EG-induced developmental toxicity EG (2500 mg/kg bw; p.o.), GA (650 mg/kg bw; p.o.), or sodium glycolate (833 mg/kg bw; s.c.; pH 7.4) were administered to time-mated pregnant CD rats to deliver high, equimolar blood glycolate levels in the presence or absence of acidosis (Carney et al., 1999). EG and GA induced a range of skeletal malformations and variations, while sodium glycolate induced only skeletal variations compared to the vehicle control group. EG also induced craniofacial defects that were not observed in GA-treated animals, but this was attributed to the almost threefold higher integrated systemic dose (area under the curve) of blood glycolate following EG administration compared to administration of GA itself.

Carney et al. (1996) further evaluated the roles of glycolate and acidosis in rat WEC. Gestation day 10.5 rat WEC were exposed for 46 hours to GA (12.5 mM; pH 6.7), sodium glycolate (12.5 mM; pH 7.4), or control medium (pH 6.7 or pH 7.4), after which time they were evaluated for growth and morphological parameters. In comparison to the pH 7.4 control, acidosis alone (i.e. pH 6.7) caused a mild impairment of embryo growth, as determined by protein content and head length, but no other measures of growth or morphogenesis were significant altered. Sodium glycolate (pH 7.4) caused a greater reduction of embryonic growth over a greater number of parameters, while also reducing overall morphological score and increasing dysmorphogenesis, primarily craniofacial lesions. GA (pH 6.7) induced an even more severe impairment of growth and morphological development.

Thus, there is evidence that developmental anomalies induced by EG is attributable to the formation and accumulation of GA in the maternal blood, though acidosis may exacerbate the induced lesions.

 

Disposition of GA into the embryo

With the role of GA and acidosis in the aetiology of EG developmental toxicity established, it is important to evaluate the disposition of GA into the embryo.

Following administration of EG (1000 mg/kg bw; p.o.) to time-mated pregnant CD rats on gestation day 11, the peak blood concentration in the dams is 4.1 mM, while in the embryo and surrounding exocoelomic fluid it is 6.3 mM and 6.7 mM respectively. Subcutaneous infusion of the same dose of EG results in respective peak GA concentrations of 0.07 mM, 0.13 mM, and 0.13 mM (Carney et al., 2011). This means that, following a teratogenic dose of EG, the concentration of GA in the rat embryo is 150% of that in the maternal blood. Following similar administration of EG (1000 mg/kg bw; p.o.) to time-mated pregnant New Zealand White rabbits on gestation day 9, the peak concentrations of GA in the maternal blood, embryo, and surrounding yolk sac cavity fluid were 1.9 mM, 0.6 mM, and 2.0 mM respectively (Carney et al., 2008). In contrast to the rat, the concentration of GA in the embryo was 30% of that in the maternal blood.

 

The disposition of GA into the rat whole embryo culture (WEC) was established by Ellis-Hutchings et al. (2014) as being predominantly mediated by a specific, energy- and pH-dependent carrier. Uptake of 14C-labelled GA from the culture medium into the embryo and yolk sac occurred against a concentration gradient over a six hour incubation period, although it reached a plateau in the embryo within two hours, such that the concentration of GA in the embryo was threefold higher than in the culture medium. The uptake of 1 mM GA was substantially and significantly reduced by incubation at low temperature (4 °C vs. 37 °C), thereby establishing energy-dependence, and enhanced under conditions of lower pH (pH 7.1 vs. pH 7.8), thereby establishing proton-dependence. Uptake of 1 mM and 12 mM GA was further inhibited by up to 80% by co-incubation with d-lactic acid (12–120 mM), suggesting that a substantial component of the uptake is mediated by a carrier protein with cross-affinity for small carboxylic acids. These characteristics have been widely described for the monocarboxylate transporter (MCT) protein family, including in the placenta of humans and rodents. This point leads to the assumption that MCT isoforms might essentially contribute to GA disposition from maternal blood to the embryo.

 

The MCT is believed to mediate the transfer of lactic acid and pyruvic acid between maternal and embryofetal compartments, although there are species differences in the characteristics of transplacental transfer. Nagai et al. (2010) examined the expression and subcellular localization of MCT isoforms in the ddY mouse placenta during gestation. Although six isoforms were initially studied, only two of these, MCT1 and MCT4, are known to be involved in the transport of monocarboxylates. The remainder were not extensively evaluated and are not discussed here. mRNA for MCT1 and MCT4 was expressed at gestation days 11, 14, 17, and 18, but not at day 9, before the development of a functional placenta. Expression was greatest early in gestation and reduced towards term. Immunohistochemical staining for the two protein isoforms showed that MCT1, which is a high-affinity transporter, is localized in the apical membrane of the syncytiotrophoblast facing the maternal blood; MCT4, which is a low-affinity transporter, is localized in the basal plasma membrane close to the embryofetal blood. A similar partitioning of MCT1 and MCT4 has been recently identified in the CD rat placenta (Moore et al., 2016).

 

In contrast to the polarity of MCT1 and MCT4 in the rodent placenta, these isoforms show the opposite polarity in placenta from non-rodent species. Settle et al. (2004) described the localisation of MCT1 and MCT4 in term human placenta. MCT1 was localised predominantly to the basal membrane (embryofetal side of the syncytiotrophoblast) while MCT4 was apparent on both plasma membranes with more intense staining on the microvillous membrane (maternal side). Moore et al. (2016) have evaluated the localisation of these two isoforms in the New Zealand White rabbit placenta. Again, MCT1 was localised on the side apposed to the embryofetal circulation while MCT4 was localised to the side of the maternal blood.

 

From these physiological differences, a model for GA disposition in rodents and non-rodents is proposed. In this model, GA is sequestered from the maternal blood and transported across the syncytiotrophoblast to the embryonic blood. In the rodent, the high affinity MCT1 apposed to the maternal blood effectively transports GA to the embryo, while MCT4 does not sequester GA from the embryonic blood as effectively. In the non-rodent, the opposite is the case. It is proposed that this qualitative difference in MCT polarity underlies the quantitative differences in maternal-embryonal disposition and response to GA. Since the polarity of the MCT in the rabbit placenta is the same as that in human, the rabbit is a more appropriate model for evaluating the intrinsic hazard relevant to humans, and therefore classification for effects upon development is not justified.

 

 

Conclusion

Given that GA disposition between the maternal blood and the embryo is driven by the polarity of MCT1 and MCT4 in the placenta, that GA is sequestered in the rat and not the rabbit, and that the rabbit and human placenta show similar polarity to each other and opposite to that of the rodent, it is proposed that the rabbit is the most appropriate species. As such, and since EG is not a developmental toxicant in the rabbit, no classification is warranted. Since read-across is considered valid at this endpoint, the conclusion of EG holds also true for EC.

 

Justification for classification or non-classification

Ethylene carbonate (EC; CAS RN 96-49-1) is not classified in the EU as a reproductive toxicant (

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008).

 

At this endpoint, read-across was used with the aim to use existing data on ethylene glycol (EG) to close data gaps without additional testing with EC. Therefore, reliable read-across data from EG are used for the risk characterization of ethylene carbonate at the endpoint developmental toxicity and fertility. The hypothesis of the analogue approach is mainly based on the assumption that EC follows the documented metabolic pathway were cyclic organic carbonates are metabolized and converted to their respective glycols.

 

According to Regulation (EC) No 1272/2008 classification of reproductive toxicants encompasses effects upon fertility and on the developing offspring (‘developmental toxicity’). EG does not affect fertility in animals, but developmental toxicity has been noted in some species at high doses.

 

EG causes developmental toxicity—characterised by craniofacial and axial-skeletal malformations and variations—in mice and rats when administered by oral gavage during the period of organogenesis. In contrast, the administration of EG to pregnant rabbits during organogenesis at doses up to 2000 mg/kg bw/d by oral gavage had no effect upon the progeny, while the highest dose was associated with substantial (42%) maternal mortality.

 

Subsequent investigations, bothin vivo and in vitro, have established that the developmental toxicity of EG in rats is related to the accumulation of glycolic acid (GA) in the blood and metabolic acidosis. The toxicity of GA, both in vivo and in vitro, is exacerbated under acidic conditions and is related to disposition. When EG was administered to rats and rabbits at a developmentally toxic dose (1000 mg/kg bw, p.o.), GA was found to be preferentially distributed into the rat embryo compared to the maternal blood (embryo:blood concentration 1.54) whereas this was not the case in the rabbit (embryo:blood concentration 0.31).

 

Recent investigation demonstrated that GA uptake into the rat embryo occurs predominantly by a specific, pH-dependent, active uptake transporter protein, consistent with the proton-linked monocarboxylate transporters (MCT). Two isoforms of the MCT exist in the placenta, a high-affinity isoform (MCT1) and a low affinity isoform (MCT4). The new and unpublished results indicate that polarity of these isoforms in the mouse and rat placenta syncytiotrophoblast is opposite to that in the rabbit and human placenta. In the rodent, MCT1 lies on the side of the maternal blood, while MCT4 lies on the side of the embryonic blood; in rabbits and humans MCT1 lies on the side of the embryonic blood while MCT4 lies on the side of the maternal blood.

 

Given that GA disposition between the maternal blood and the embryo is driven by the polarity of MCT1 and MCT4 in the placenta, that GA is sequestered in the rat and not the rabbit, and that the rabbit and human placenta show similar polarity to each other and opposite to that of the rodent, it is proposed that the rabbit is the most appropriate species. As such, and since EG is not a developmental toxicant in the rabbit, no classification is warranted under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/218. Since read-across is considered valid at this endpoint, the conclusion of EG holds also true for EC.