Bis(2-ethylhexyl) carbonate

Endpoint summary

Description of key information

The most critical NOAEL of 250 mg/kg bw/d was obtained in the 90 d repeated dose toxicity study in rats according to OECD guideline 408 with the read-across substance 2-Ethylhexanol (due to longer study duration). 
In a 28 d study according to OECD guideline 407 conducted with Bis(2-ethylhexyl) carbonate, a NOAEL of 1000 mg/kg bw/d was obtained.
Recalculation based on molecular weight (2-Ethylhexanol: 130.23 g/mol; Bis(2-ethylhexyl) carbonate: 286.45 g/mol) and the release of 2 moles of 2-Ethylhexanol per mole of Bis(2-ethylhexyl) carbonate resulted in a NOAEL of 275 mg/kg bw/d for Bis(2-ethylhexyl) carbonate (although the amount of the  2-ethylhexanol moiety in Bis(2 -ethylhexyl) carbonate is 286.45 mg/mol, for recalculating the NOAEL of 2-Ethylhexanol to  Bis(2-ethylhexyl)carbonate twice the molecular weight of 2-Ethylhexanol (260.46 mg/mol) was used).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Read-across from GLP-guideline study. In accordance to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 42-43 days
- Weight at study initiation: 105-114 g (males); 86-97 g (females)
- Fasting period before study: no data
- Housing: singly in stainless steel cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Doses were prepared daily by dispersing 2EH in an aqueous solution of Cremophor EL (5 µg/100 ml) by ultra high speed sonication for 1 min.
Homogeneity was maintained by magnetic stirring throughout dosing.


VEHICLE
- Justification for use and choice of vehicle (if other than water): a surfactant (Cremophor) was used to facilitate mixing 2-EH with water
- Concentration in vehicle: 5 µg/100 ml
- Amount of vehicle (if gavage): dose volume was 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations and homogeneity were checked by gas chromatographic analysis of samples from each dose level at the start of the preliminary 11 -day studies and periodically in 13-week studies.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

5/week

Remarks:

Doses / Concentrations:
0, 25, 125, 250, 500mg/kg bw /day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of preliminary 11-day studies
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: determination of peroxisome prolieferation (3 animals per dose level; Table [4])
- Post-exposure recovery period in satellite groups: none
- Section schedule rationale (if not random): random

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected twice daily for morbidity or mortality, or once daily on nontreatment days. Clinical observations were
made daily.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at start and thereafter at weekly intervals


BODY WEIGHT: Yes
- Time schedule for examinations: at start and thereafter at weekly intervals


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n.a.


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 data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations:


OPHTHALMOSCOPIC EXAMINATION: No data
- Time schedule for examinations:



HAEMATOLOGY: Yes
- Time schedule for collection of blood: on study days 29 and 84.
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: not specified
- Parameters examined: leucocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular
hemoglobin, mean corpuscular hemoglobin concentration, platelets and differential leucocytes, and reticulocytes


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on study days 29 and 84.
- Animals fasted: No data
- How many animals: not specified
- Parameters checked in table [1] were examined.


URINALYSIS: No
- Time schedule for collection of urine: n.a.
- Metabolism cages used for collection of urine: No
- Animals fasted: No data



NEUROBEHAVIOURAL EXAMINATION: No data



OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Adrenals, brains, kidneys, livers, stomachs, testes, and ovaries from all animals were weighed, and with other
organs and tissues listed in U.S. EPA Health Effects Guidelines (1987b) fixed in 4% formalin.
HISTOPATHOLOGY: Yes. All tissues from high dose and control animals were stained with hematoxylin—eosin and examined microscopically.
Lungs, livers (including gallbladders in mice), spleens, kidneys, stomachs, sternums, femurs, and femur bone marrows were examined microscopically at intermediate dose levels.
Skin, eyes, female mammary glands, thigh musculatures, and extraorbital lacrymatory glands were not examined in the absence of signs of toxicity.
Livers were also stained with oil red for lipid content and examined microscopically.

Other examinations:

Hepatic peroxisome proliferation: livers were removed at termination and weighed, and cyanide-insensitive pCoA
activities (Lazarow, 1981) and protein concentrations (Lowry et al, 1951) were determined (week 13; ancillary group; 3 animals per dose level)

Statistics:

Means and standard deviations were calculated for body weights, food and water consumption, clinical pathology results, and organ weights.
Values for test groups were compared with controls in the main study by ANOVA followed by Dunnett's test (Dunnett, 1955, 1964)
and in ancillary studies by ANOVA followed by Student's t test (Winer, 1971).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities or clinical findings differing from controls at any treatment level.

BODY WEIGHT AND WEIGHT GAIN
There was decreased weight gain in male and female rats at 500 mg/kg, starting at Week 4 in males and Week 11 in females,
amounting to weight losses of 7% in males and 6% in females at termination (both p<0.01).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
n.a.

FOOD EFFICIENCY
n.a.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
n.a.

OPHTHALMOSCOPIC EXAMINATION
n.a.

HAEMATOLOGY
There was a 25% increase in reticulocyte numbers in male and female rats at 500 mg/kg in Week 13.

CLINICAL CHEMISTRY
Differences from controls rats were seen mostly at 84 days (data not shown).
Males at 500 mg/kg/day: 13% decreases in total protein and albumin concentrations.
Females at 250 mg/kg/day: 30 % decrease in serum ALT activities
Females at 500 mg/kg/day: 36% decreases in serum ALT activities;
16% decrease in serum cholesterol concentration

URINALYSIS
n.a.

NEUROBEHAVIOUR
n.a.

ORGAN WEIGHTS
Relative organ weights: significant differences from control rats were moderate and limited to the brain, kidneys, liver, stomach, and testes at 250 and 500 mg/kg. Organ weights were increased in both male and female rats (cf. table).


Table: Relative organ weights (a) in rats at termination of the 13-Week oral gavage rat study (b)

Males [Dose (mg/kg bw/day)] Females [Dose (mg/kg bw/day)]
0 250 500 0 250 500
--------------------------------------------------------------------
Brain 0.68 0.70 0.72** 1.07 1.1 1.1
Kidneys 0.69 0.75** 0.81** 0.77 0.81* 0.82**
Liver 2.77 2.98** 3.57** 2.67 2.88** 3.07**
Stomach 0.57 0.58 0.63** 0.71 0.75* 0.82**
Testes/Ovaries 1.11 1.16 1.17* 0.041 0.037* 0.039
--------------------------------------------------------------------------------------
values are mean organ/body weight ratios
there were no significant differences from controls in rats at 25 and 125 mg/kg bw/day
* p<0.05; ** p<0.01



GROSS PATHOLOGY
Gross lesions differing from controls in both species were seen at 500 mg/kg bw/day only. In rats 2/10 males and 4/10 females exhibited single
or multiple slightly elevated foci in the forestomach.

HISTOPATHOLOGY: NON-NEOPLASTIC
Dose-related findings in rats (data not shown) were limited to the forestomach and liver at 500 mg/kg.
Forestomach: there was a generalized acanthosis of the forestomach mucosa in 1/10 males with ballooning degeneration of the epithelial wall and acanthosis of the forestomach mucosa in 2/10 males and 5/10 females.
Liver: there was a moderate decrease in hepatic peripheral lobular fatty infiltration in 4/10 males and 2/10 females and adrenal ß-cell hyperplasia in 3/10 female rats.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
n.a.

HISTORICAL CONTROL DATA (if applicable)
n.a.

OTHER FINDINGS
Peroxisome proliferation:
In Week 13, increases in pCoA activity were 6.5-fold in male rats (p<0.001) and 3.4-fold in females (p<0.05) at 500 mg/kg . Reportedly, decreases in body weight gain were similar to those in the main study animals.

--------------------------------------------------------------------------------------
Dose (mg/kg bw/day)
0 25 125 250 500
--------------------------------------------------------------------
Mean PCoA activity (nanomol/min/mg of protein)
Males 3.38 4.49 5.21 6.24 22.0*
Females 2.95 4.54 5.01 6.6 9.92**
--------------------------------------------------------------------------------------
*p<0.001 and **p<0.05 by ANOVA/Students ttest

Dose descriptor:

NOEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: absence of treatment-related effects on target organs

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: absence of treatment-related systemic effects on target organs

Critical effects observed:

not specified

Table: Relative organ weights (a) in rats at termination of the 13-Week 
oral gavage rat study (b)

	Males [Dose (mg/kg bw/day)]			Females [Dose (mg/kg bw/day)]
	0	250	500	0	250	500
Brain	0.68	0.70	0.72**	1.07	1.1	1.1
Kidneys	0.69	0.75**	0.81**	0.77	0.81*	0.82**
Liver	2.77	2.98**	3.57**	2.67	2.88**	3.07**
Stomach	0.57	0.58	0.63**	0.71	0.75*	0.82**
Testes/Ovaries	1.11	1.16	1.17*	0.041	0.037*	0.039

(a) values are mean organ/body weight ratios

(b) there were no significant differences from controls in rats at 25 and 125 mg/kg bw/day

* p0.05; ** p0.01    

Conclusions:

Valid subchronic oral gavage rat study.
(1) 2-EH was moderately toxic at all dose levels up to and including 500 mg/kg bw/day; based
- on lack of mortality and clinical signs;
- moderately reduced body weights in groups at 500 mg/kg bw/day;
- minor effects of low relevance on clinicochemcial and hematological parameters
(2) Target organs were the liver, forestomach, and the kidneys; based on significantly increased relative organ weights at termination
(3) Local irritating effects prevailed; systemic effects were low; based on
- inflammation in the forestomach,
- and lack of treatment-related findings in other organs, including testes
(4) 2-EH induces peroxisome proliferation in rats; observed at 500 mg/kg bw/day in rats of both sexes
(5) The NOEL (no observable effect level) was 125 mg/kg bw/day. A NOAEL (no observable adverse effect level) was not derived, but may be estimated to be 250 mg/kg bw/day, based on the above

Executive summary:

The subchronic effects of 2 -EH were studied in a 90-day oral gavage study using male and female rats (10 animals per sex and dose). The test dose levels were 0, 25, 125, 250, and 500 mg/kg bw/day, based on the results of preliminary 11-day studies.

Key results include:

• Mortalities or clinical findings were not different from controls
• Food consumption was comparable to controls
• Body weight gain was reduced in the high dose groups, resulting in decreased terminal weights in males (-7%) and females (-6%); (both p<0.01)
• Clinicochemical changes were seen in high dose groups (males:total protein and albumin -13%; females: serum cholesterol -16%); biological significance is unclear
• Hematology: reticulocytes were increased (25%) in high dose males and females
• Organ weights: target organs were liver, forestomach, and kidneys; based on increased relative weights (p<0.01) in male and female groups at 250 and 500 mg/kg bw/day. No weight changes were noted at 25 and 125 mg/kg bw/day.
• Reproductive organs: relative weight of testes was increased, and that of ovaries decreased, at 250 and 500 mg/kg bw/day.
• Histopathology revealed changes only in high dose animals. Predominantly inflammatory changes in the forestomach which are attributable to the irritation properties of 2 -EH
• 2 -EH at 500 mg/kg bw/day caused peroxisome proliferation, as evidenced by a statistically significant increase of the hepatic cyanide-insensitive palmitoyl coenzyme A activity in male (p<0.001) and female (p<0.05) rats in Week 13.
• The subchronic NOEL was 125 mg/kg bw/day in male and female rats. The estimated NOAEL is 250 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

275 mg/kg bw/day

Study duration:

subchronic

Species:

rat

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

Additional information

For the assessment of the repeated dose toxicity of Bis(2-ethylhexyl) carbonate a 14 day dose range finding study as well as a full 28 d oral repeated dose toxicity guideline study are available.

In a 14-day Oral Repeated Dose Range Finding Study according OECD Guideline 407 and Directive 96/54 EEC B.7 with Bis(2-ethylhexyl) carbonate (99 -100% a.i.) no clear specific compound related effects were observed.

The test item was orally administered in graduated doses to 5 groups of male and female wistar rats by gavage, using a stomach tube. One group, receiving the vehicle cotton seed oil, served as control. In total, 14 applications per animal were administered.

The following doses were evaluated: 0 (vehicle control), 50, 250, 500, 1000, and 2000 mg/kg bw/day. The volume of application was 5 mL/kg bw.

All rats survived throughout the test period without showing relevant clinical-toxic effects and were sacrificed on day 14. No significant differences in weight gain between the dosed groups could be detected. All animals showed normal food intake and no obvious reduction in food consumption was found. Most evaluated haematology and clinical biochemistry-values were within the expected ranges. Single deviations for individual values were found throughout all groups. No dose dependency can be concluded. Considering the reported data the following doses were recommended for the 28 day Toxicity study: 0 (vehicle control), 150, 500, and 1000 mg/kg bw/day.

In a Repeated Dose, 28-day Oral Toxicity Study performed according to First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No.407,"Repeated Dose 28-day Oral Toxicity Study in Rodents" adopted 27 July, 1995 and Directive 96/514 EEC B.7., Bis(2-ethylhexyl) carbonate (99 -100 % a.i.) suspended in cotton seed oil as vehicle was orally administered in graduated doses of 150, 500 and 1000 mg/kg bw/day to groups of 5 male and 5 female wistar rats by gavage, using a stomach tube. A total of 28 applications per animal were administered. All rats treated with the test item survived throughout the test period without showing any clinical-toxic effects and were sacrificed on day 28. No differences were observed concerning functional and behavioural examination prior to application and during the last week of dosing, respectively. The mean weight gain in all groups was almost as high, as expected according to the standard growth curve for this strain. Individually diminished weight gains are due to the daily treatment associated with mechanical manipulation and therefore with increased stress for the animals. All animals showed normal food intake and no significant reduction in food consumption was found. No relevant changes were recorded upon necropsy. Except of organ weights and histopathological findings in male rats of all dose groups indicative for a alpha2µ globulin nephropathy, which is a male rat-specific phenomenon and not considered being relevant to man, no substance related relevant changes in organ weights or histopathology were found. Also, the hematological, clinical chemical and urine parameters were without findings of toxicological relevance. Therefore, no 'no observed effect dose level' (NOEL) of Bis(2-ethylhexyl) carbonate suspended in cotton seed oil and administered in a total of 28 applications by gavage over a period of 28 days could be found in consideration of the kidney changes found in all dosed male animals. However, the 'no observed adverse effect dose level' (NOAEL) of Bis(2-ethylhexyl) carbonate is considered to be 1000 mg/kg bw as the most dominant effect, the kidney droplets combined with statistically increased kidney weights is strongly indicative of hyaline droplet (alpha2µ globulin) nephropathy. As the protein alpha2µ globulin is prominent in male rats but present only in traces in humans, this mechanism of pathogenesis and the associated pathological findings are generally not considered relevant to safety assessment for man.

 

90 day repeated dose toxicity studies conducted in rats and in mice are available for the read-across substance 2-Ethylhexanol. The read-across approach is justified based on metabolism.

As Bis(2-ethylhexyl) carbonate does not penetrate the skin (c.f. IUCLID entry Dermal absorption: 14858-73-2_8.8.1_THG_2007_OECD 428) and has a very low vapour pressure and a use pattern which will not generate inhalative exposure, the only route of exposure relevant for risk assessment is oral ingestion.

The organic branched chain carbonate Bis(2-ethylhexyl) carbonate is expected to be acid labile and thus will break down to its carbonate and alcohol moieties at contact with the acidic gastric juice. Hydrolysis has been demonstrated in an in vitro study.

The resulting carbonic acid, respectively the carbonate anion and carbon dioxide are uncritical from a toxicological view due to natural regulation mechanisms.

The released alcohol moiety 2-Ethylhexanol might be systemically absorbed and thus might have a systemic effect and is therefore the relevant substance for read across. To justify this read across approach, the postulated gastric break down was experimentally verified in an in vitro hydrolysis study. The results of this hydrolysis study showed a hydrolysis of Bis(2-ethylhexyl) carbonate of about 65% within 4 hours ingastric-fluid simulant and a hydrolysis of about 15% within 4 hours in intestinal-fluid simulant. Enzymes capable of hydrolysing Bis(2-ethylhexyl) carbonate are present in the gastrointestinal tract, and thus Bis(2-ethylhexyl) carbonate will start to be metabolised prior to absorption.

The subchronic effects of 2 -EH were studied in a 90-day oral gavage study using male and female rats (10 animals per sex and dose). The test dose levels were 0, 25, 125, 250, and 500 mg/kg bw/day, based on the results of preliminary 11-day studies.

Key results include:

- Mortalities or clinical findings were not different from controls

- Food consumption was comparable to controls

- Body weight gain was reduced in the high dose groups, resulting in decreased terminal weights in males (-7%) and females (-6%); (both p<0.01)

- Clinicochemical changes were seen in high dose groups (males:total protein and albumin -13%; females: serum cholesterol -16%); biological significance is unclear

- Hematology: reticulocytes were increased (25%) in high dose males and females

- Organ weights: target organs were liver, forestomach, and kidneys; based on increased relative weights (p<0.01) in male and female groups at 250 and 500 mg/kg bw/day. No weight changes were noted at 25 and 125 mg/kg bw/day.

- Reproductive organs: relative weight of testes was increased, and that of ovaries decreased, at 250 and 500 mg/kg bw/day.

- Histopathology revealed changes only in high dose animals. Predominantly inflammatory changes in the forestomach which are attributable to the irritation properties of 2 -EH

- 2 -EH at 500 mg/kg bw/day caused peroxisome proliferation, as evidenced by a statistically significant increase of the hepatic cyanide-insensitive palmitoyl coenzyme A activity in male (p<0.001) and female (p<0.05) rats in Week 13.

The subchronic NOEL was 125 mg/kg bw/day based on minor organ weight changes in male and female rats. The estimated NOAEL is 250 mg/kg bw/day.

 

The subchronic effects of 2 -EH were studied in a 90-day oral gavage study using male and female B6C3F1 mice (10 animals per sex and dose). The test dose levels were 0, 25, 125, 250, and 500 mg/kg bw/day, based on the results of preliminary 11-day studies.

Key results include:

- there were no mortalities or clinical findings differing from controls

- food consumption was comparable to controls

- body weight gain was comparable to controls

- there were no clinicochemical or hematological changes at any dose level

- Organ weight changes: target organs were liver and forestomach; a dose-related increase of relative weight was seen in males and females; a level of statistical significance (p<0.05) was gained in males at 250 and 500 mg/kg bw/day

- Male and female reproductive organs: no weight change noted

- Histopathology revealed a low incidence of inflammatory changes only in high dose animals; regarded to be incidental; possibly attributable to the irritation properties of 2 -EH

- 2 -EH was not a peroxisome proliferator in B6C3F1 mice at up to and including 500 mg/kg bw/day

 The subchronic NOEL was 125 mg/kg bw/day based on minor organ weight changes in male and female rats. The estimated NOAEL is 250 mg/kg bw/day.

There are no data gaps for the endpoint repeated dose toxicity. No human data are available.

However, there is no reason to believe that these results from rat and mouse would not be applicable to humans.

 

The most critical NOAEL of 250 mg/kg bw/d was obtained in the 90 d repeated dose toxicity study in rats with 2-Ethylhexanol.

 

Recalculation based on molecular weight (2-Ethylhexanol: 130.23 g/mol; Bis(2-ethylhexyl) carbonate: 286.45 g/mol) and the release of 2 moles of 2-Ethylhexanol per mole of Bis(2-ethylhexyl) carbonate resulted in a NOAEL of 275 mg/kg bw/d for Bis(2-ethylhexyl) carbonate. Although the amount of the 2-ethylhexanol moiety in Bis(2 -ethylhexyl) carbonate is 286.45 mg/mol, for recalculating the NOAEL of 2-Ethylhexanol to Bis(2-ethylhexyl)carbonate twice the molecular weight of 2-Ethylhexanol (260.46 mg/mol) was used.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
OECD guideline 408 study, no deviations, GLP - this study was selected due to longer study duration

Justification for classification or non-classification

Based on the available data, Bis(2-ethylhexyl) carbonate does not need to be classified for repeated dose toxicity according to the criteria given in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC. Thus, no labelling is required.