Glycerides, C16-18 and C18-unsatd. mono-, di- and tri-, citrates, potassium salts

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A 90 day oral feeding study with the structural analogue Castor oil was performed equivalent to OECD Guideline 408 both in F344/N rats and B6C3F1 mice (Irwin, NTP report 1992). The test substance was mixed at concentrations of 0, 0.62, 1.25, 2.50, 5.00, 10.0 % (w/w) to the diet and the animals (10/sex/concentration) were fed ad libitum for 13 weeks. The highest dose was equivalent to approx. 5.7 g/kg bw/day for rats and approx. 15 g/kg bw/day for mice. No matings were performed, but male and female fertility parameters were analyzed in rats and mice including oestrous cycle length, caudal weight, epididymal weight, testis weight, sperm count/g testis, sperm motility (%) and histopathology of organs relevant for reproduction (including adrenal glands, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, mammary gland, pituitary gland, preputial or clitoral glands). A complete histopathologic examination was conducted on all rats and mice from the control and 10% dose groups.

No significant changes were noted in a screening study for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of oestrous cycles of rats or mice given diets containing castor oil. No histopathologic abnormalities were found in the reproductive organs.

A NOAEL of 5000 mg/kg bw/day for rats and a NOAEL of 15000 mg/kg bw/day for mice could be identified based on parental fertility parameters.

Link to relevant study records

Reference

Reference 1

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 408

Principles of method if other than guideline:

Within a 90 day oral feeding study performed equivalent to OECD guideline 408 with Castor oil, male and female fertility parameters were analyzed in mice. No matings were performed.

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA, USA)
- Age at study initiation: 6 weeks
- Weight at study initiation: male mice: 22.6 - 23.0 g, female mice: 17.2 - 17.7 g
- Housing: individually in Polycarbonate cages lined with heat-treated hardwood chips, covered with polyester filter sheets.
- Diet (e.g. ad libitum): Control feed (NIH 07) or diet formulations of castor oil were available ad libitum; feeders were changed twice per week throughout the study.
- Water (e.g. ad libitum): automatic watering system
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76°F
- Humidity (%): 42% to 72%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amount of feed in a twin-shell blender and blended for 15 minutes to achieve a uniform mix. The homogeneity of castor oil in feed at 10% (100 mg/g) was determined by gravimetric analysis, and blends at 0.5% (5 mg/g) were determined by HPLC analysis. These concentrations of chemical in feed were found to be homogeneously distributed by this mixing procedure. The stability of the 0.5% dose level was determined using HPLC; it was found to be stable for at least 21 days when stored in the dark at 5°C and for 3 days when stored open to air and light in a rodent cage. During the studies, formulated diets were stored for no longer than 3 weeks at 5°C; feed hoppers in the animal cages were changed twice weekly.

Details on mating procedure:

no matings performed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analyses for all dose mixtures given to the animals ranged from 97% to 106% of the target concentrations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily ad libitum feeding

Dose / conc.:

0 other: %

Dose / conc.:

0.62 other: %

Remarks:

Equivalent to 917 mg compound consumed/kg bw/day (males)
Equivalent to 1153 mg compound consumed/kg bw/day (females)

Dose / conc.:

1.25 other: %

Remarks:

Equivalent to 2022 mg compound consumed/kg bw/day (males)
Equivalent to 2282 mg compound consumed/kg bw/day (females)

Dose / conc.:

2.5 other: %

Remarks:

Equivalent to 3800 mg compound consumed/kg bw/day (males)
Equivalent to 5009 mg compound consumed/kg bw/day (females)

Dose / conc.:

5 other: %

Remarks:

Equivalent to 7823 mg compound consumed/kg bw/day (males)
Equivalent to 9627 mg compound consumed/kg bw/day (females)

Dose / conc.:

10 other: %

Remarks:

Equivalent to 15017 mg compound consumed/kg bw/day (males)
Equivalent to 16786 mg compound consumed/kg bw/day (females)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/ CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: initially and 1 x wk thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

Proestrus, Estrous, Metestrous, Diestrous, Cycle Length (days)

Sperm parameters (parental animals):

Caudal weight, epididymal weight, testis weight, Sperm count/g testis, Sperm motility (%)

Litter observations:

not performed

Postmortem examinations (parental animals):

Complete histopathology examinations were conducted on all mice from the control and 10% dose groups. Organ weights were determined to the nearest milligram for the liver, right kidney, right testicle, heart, thymus, and lungs. All tissues were preserved in 10% neutral buffered formalin.

The following tissues were routinely processed for preparation of histologic sections and microscopic examination: adrenal
glands, brain, cecum, colon, duodenum, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, esophagus, eyes (if
grossly abnormal), femur (including marrow), heart, ileum, jejunum, kidneys, liver, lungs and mainstem bronchi, mammary
gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland,
preputial or clitoral glands, rectum, salivary glands, skin, spinal cord and sciatic nerve (if neurologic signs present), spleen,
forestomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions
and tissue masses including regional lymph nodes. A complete histopathologic examination was conducted on all rats and
mice from the control and 10% dose groups. Liver was examined from male rats in all other dose groups, and histologic
sections of gross lesions were examined from all rats.

Postmortem examinations (offspring):

not performed

Statistics:

Dunn's test; Shirley's test.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights of rats receiving diets containing castor oil did not differ significantly from controls.
Mean body weights of exposed male mice generally were lower than controls, while mean body weights of exposed females generally were higher. There were no obvious indications that these differences were related to dietary concentrations of castor oil, except that mean body weights of male mice receiving the 10% castor oil diet were consistently lower than those of control mice from week 3 through the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption by female mice receiving diets containing 10% castor oil was slightly lower than controls.

ORGAN WEIGHTS
Castor oil exposure produced no adverse effects on any male (testes weight, epididymal sperm motility, density, or testicular spermatid head count) or female (estrual cycle length, or time spent in each phase of the cycle) reproductive parameter among mice. The low value for sperm motility in control mice was attributed to poor preparative technique.

Key result

Dose descriptor:

NOAEL

Remarks:

parental fertility parameters

Effect level:

ca. 15 000 mg/kg bw/day (actual dose received)

Based on:

other: calculated test material intake based on food consumption and body weight

Sex:

male/female

Basis for effect level:

other: for mice based on oestrus stage and cycle length and sperm characterization.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

not examined

Key result

Dose descriptor:

other: No F1 generation

Basis for effect level:

other: No F1 generation

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Table 1: Reproductive System Data for B6C3F1 Mice in the 13-Week Feed Studies

of Castor Oil

 

	Percent in Feed
	0	2.5	5	10
Male^a
Left caudal weight (mg)	15	13	16	16
Left epididymal weight (mg)	45	46	46	44
Left testis (mg)	121	120	121	119
Sperm count (x106)/g testis	179.2	162.4	170.1	158.3
Sperm motility (%)	39.2	53.7	45.4	52.2
Female^b
Estrous stage (%)
Proestrus	12.5	14.2	15.8	16.7
Estrous	28.3	32.5	25.8	25.8
Metestrous	18.3	19.2	18.3	19.2
Diestrous	40.8	34.2	39.2	38.3
Not clear or no cells observed	0.0	0.0	0.8	0.0
Cycle Length (days)	5.0	5.1	5.2	5.1

^aMean for groups of 10 animals; no significant difference vs. the controls by Dunn's test

^bMean for groups of 10 animals unless otherwise specified

 

Conclusions:

A NOAEL of 15000 mg/kg bw/day for mice could be identified based on parental fertility parameters.
Matings were not performed in this study.

Executive summary:

In a subchronic toxicity study castor oil (CAS 8001-79-4) was administered to 10 mice/sex/dose in the diet at dose levels up to 10%.

To screen for potential reproductive toxicity, sperm motility and morphology were evaluated at necropsy, and vaginal cytology was evaluated on core-study animals during the week just preceding necropsy. For the 12 days prior to termination, females were subject to vaginal lavage with saline. The relative preponderance of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were used to identify the stages of the estrual cycle. Sperm motility and sperm density was evaluated at necropsy, as well as the number of spermatid heads per total testis and per gram of testis. No adverse effects have been reported at any dose. The NOAEL is 15000 mg/kg bw/d (the highest dose employed).

This subchronic toxicity study in the rat/mouse is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408).

Reference 2

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 408

Principles of method if other than guideline:

Within a 90 day oral feeding study performed equivalent to OECD guideline 408 with Castor oil, male and female fertility parameters were analyzed in rats. No matings were performed.

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
- Source: Simonsen Laboratories (Gilroy, CA, USA)
- Age at study initiation: 6 weeks
- Weight at study initiation: male rats: 126 - 132 g; female rats: 107- 110 g
- Housing: rats: 5 per cage, Polycarbonate cages lined with heat-treated hardwood chips, covered with polyester filter sheets.
- Diet (e.g. ad libitum): Control feed (NIH 07) or diet formulations of castor oil were available ad libitum; feeders were changed twice per week throughout the study.
- Water (e.g. ad libitum): automatic watering system
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76°F
- Humidity (%): 42% to 72%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amount of feed in a twin-shell blender and blended for 15 minutes to achieve a uniform mix. The homogeneity of castor oil in feed at 10% (100 mg/g) was determined by gravimetric analysis, and blends at 0.5% (5 mg/g) were determined by HPLC analysis. These concentrations of chemical in feed were found to be homogeneously distributed by this mixing procedure. The stability of the 0.5% dose level was determined using HPLC; it was found to be stable for at least 21 days when stored in the dark at 5°C and for 3 days when stored open to air and light in a rodent cage. During the studies, formulated diets were stored for no longer than 3 weeks at 5°C; feed hoppers in the animal cages were changed twice weekly.

Details on mating procedure:

no matings performed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analyses for all dose mixtures given to the animals ranged from 97% to 106% of the target concentrations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily ad libitum feeding

Dose / conc.:

0 other: %

Dose / conc.:

0.62 other: %

Remarks:

Equivalent to 404 mg compound consumed/kg bw/day (males)
Equivalent to 401 mg compound consumed/kg bw/day (females)

Dose / conc.:

1.25 other: %

Remarks:

Equivalent to 809 mg compound consumed/kg bw/day (males)
Equivalent to 797 mg compound consumed/kg bw/day (females)

Dose / conc.:

2.5 other: %

Remarks:

Equivalent to 1583 mg compound consumed/kg bw/day (males)
Equivalent to 1569 mg compound consumed/kg bw/day (females)

Dose / conc.:

5 other: %

Remarks:

Equivalent to 3067 mg compound consumed/kg bw/day (males)
Equivalent to 3045 mg compound consumed/kg bw/day (females)

Dose / conc.:

10 other: %

Remarks:

Equivalent to 5835 mg compound consumed/kg bw/day (males)
Equivalent to 5725 mg compound consumed/kg bw/day (females)

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/ CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: initially and 1 x wk thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

Proestrus, Estrous, Metestrous, Diestrous, Cycle Length (days)

Sperm parameters (parental animals):

Caudal weight, epididymal weight, testis weight, Sperm count/g testis, Sperm motility (%)

Litter observations:

not performed

Postmortem examinations (parental animals):

Complete histopathology examinations were conducted on all rats from the control and 10% dose groups. Livers were examined from male rats in all other dose groups; histologic sections of gross lesions were examined from all rats. Organ weights were determined to the nearest milligram for the liver, right kidney, right testicle, heart, thymus, and lungs. All tissues were preserved in 10% neutral buffered formalin.

The following tissues were routinely processed for preparation of histologic sections and microscopic examination: adrenal glands, brain, cecum, colon, duodenum, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, esophagus, eyes (if grossly abnormal), femur (including marrow), heart, ileum, jejunum, kidne s, liver, lungs and mainstem bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituita y gland, preputial or clitoral glands, rectum, salivary glands, skin, spinal cord and sciatic nerve (if neurologic signs present), spleen, forestomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions and tissue masses including regional lymph nodes. A complete histopathologic examination was conducted on all rats from the control and 10% dose groups. Liver was examined from male rats in all other dose groups, and histo ogic sections of gross lesions were examined from all rats.

Postmortem examinations (offspring):

not performed

Statistics:

Dunn's test; Shirley's test.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights of rats receiving diets containing castor oil did not differ significantly from controls. Mean body weights of exposed female rats were slightly lower than the mean body weights of controls but the differences were not dose-related.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant differences in average food consumption among each sex were observed, although food consumption of male and female rats receiving diets containing 10% castor oil was slightly lower than that of controls.

ORGAN WEIGHTS
In male rats, there was a slight decrease in epididymal weight (6-7%) which occurred in the middle- and high-dose groups, but this was not dose-related. There were no effects on any other male rat reproductive endpoint, or on any female rat reproductive endpoint. Although there was some variation in epididymal weights, their small magnitude and the absence of changes in other endpoints suggested that there was little or no evidence of any reproductive toxicity associated with castor oil exposure. Histopathologic examination revealed an absence of compound-related lesions in any organ or tissue of rats exposed to castor oil in the diet.

Key result

Dose descriptor:

NOAEL

Remarks:

parental fertility parameters

Effect level:

ca. 5 000 mg/kg bw/day (actual dose received)

Based on:

other: calculated test material intake based on food consumption and body weight

Sex:

male/female

Basis for effect level:

other: for rats based on oestrus stage and cycle length and sperm characterization.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

not examined

Key result

Dose descriptor:

other:

Remarks:

No F1 generation.

Basis for effect level:

other: No F1 generation.

Remarks on result:

not measured/tested

Reproductive effects observed:

not specified

Table 1: Reproductive System Data for F344/N Rats in the 13-Week Feed Studies

of Castor Oil

 

	Percent in Feed
	0	2.5	5	10
Male^a
Left caudal weight (mg)	151	153	145	153
Left epididymal weight (mg)	502	498	464*	476
Left testis (mg)	1539	1550	1463	1492
Sperm count (x10^6)/g testis	72.8	65.9	71.7	77.5
Sperm motility (%)	73.6	65.9	72.1	69.8
Female^b
Estrous stage (%)
Proestrus	12.5	14.2	15.8	16.7
Estrous	28.3	32.5	25.8	25.8
Metestrous	18.3	19.2	18.3	19.2
Diestrous	40.8	34.2	39.2	38.3
Not clear or no cells observed	0.0	0.0	0.8	0.0
Cycle Length (days)	5.0	5.1	5.2	5.1

^aMean for groups of 10 animals; no significant difference vs. the controls by Dunn's test

^bMean for groups of 10 animals unless otherwise specified

* Significantly different from control groups by Shirley's test ; p < 0.05.

 

Conclusions:

A NOAEL of 5000 mg/kg bw/day for rats could be identified based on parental fertility parameters.
Matings were not performed in this study.

Executive summary:

In a subchronic toxicity study castor oil (CAS 8001-79-4) was administered to 10 rats/sex/dose in the diet at dose levels up to 10%.

To screen for potential reproductive toxicity, sperm motility and morphology were evaluated at necropsy, and vaginal cytology was evaluated on core-study animals during the week just preceding necropsy. For the 12 days prior to termination, females were subject to vaginal lavage with saline. The relative preponderance of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were used to identify the stages of the estrual cycle. Sperm motility and sperm density was evaluated at necropsy, as well as the number of spermatid heads per total testis and per gram of testis. No adverse effects have been reported at any dose. The NOAEL is 5000 mg/kg bw/d (the highest dose employed).

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirement for a subchronic oral study (OECD 408).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Sufficient to address requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In a developmental toxicity study the structural analogue medium chain triglycerides (MCT) was administered to Crl:CD BR rats by intravenous infusion at dose levels of 1000 and 4280 mg/kg bw/day from days 6 through 15 of gestation.The maternal LOAEL is 4280 mg/kg bw/day, based on an increasing incidence of necropsy findings, including enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. The maternal NOAEL is 1000 mg/kg bw/day. No adverse effects related to prenatal development have been reported. The developmental NOAEL is 4280 mg/kg bw/day.

In a developmental toxicity study medium chain triglycerides (MCT) was administered to females Hra:(NZW)SPF rabbits (15/dose) by intravenous infusion at dose levels of 1000, and 4280 mg/kg bw/day from days 7 through 19 of gestation. The maternal LOAEL is 4280 mg/kg bw/day, based on strongly reduced food consumption, significant loss of body weight, no faecal output in some animals.The maternal NOAEL is 1000 mg/kg bw/day. The developmental NOAEL is 4280 mg/kg bw/day. Increased resorptions, decreased foetal body weights, and increased incidence of morphological anomalies are reported at 4280 mg/kg bw/d. These foetal effects were due to the result of dietary deprivation, maternal toxicity, or both, rather than a direct teratogenic effect of the test article.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

parenteral route of application, only two dose levels, only two dose levels, number of corpora lutea not reported

Principles of method if other than guideline:

In this study, we evaluate the developmental toxicity of a 20% lipid emulsion that contains a 3:1 ratio of medium chain triglyceride (MCT) to one long chain containing lipid emulsion (LCT).

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

other: Hra:(NZW)SPF rabbits

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HRP, Inc. (Denver, PA)
- Age at study initiation: 5.5 to 6.5 months
- Weight at study initiation: 3300 to 4446 g on GD 0
- Housing: individually in suspended stainless steel cages
- Diet (e.g. ad libitum): ad libitum except during dose administration
- Water (e.g. ad libitum): ad libitum except during dose administration

ENVIRONMENTAL CONDITIONS
Environmental controls in the animal rooms were set to maintain temperature, relative humidity, and light/dark cycle

Route of administration:

intravenous

Vehicle:

other: 20% lipid emulsion was created, no further specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose was administered daily to rats by intravenous infusion via a caudal vein using a Quik-Cath Teflon catheter connected to a syringe using an extension set. The dose was administered daily to rabbits via a marginal ear vein using an indwelling catheter [Insyte-W (24-gauge, 3/4-inch intravenous catheter and needle unit attached to a 30-inch extension set, Abbott Laboratories)]. Doses for rats and rabbits were delivered using syringe pumps. The rabbits were acclimated to the dose restraint apparatus for 2.5 and 5 h on the 2 days preceding initiation of dosing

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Mated rabbits were received on GD 2 or 3 in two consecutive weekly shipments and were observed at arrival for abnormalities indicative of health problems.

Duration of treatment / exposure:

GD 7 through 19

Frequency of treatment:

daily, approximately 5 h/day
A 4- or 5-h infusion was used to approximate longer-term infusion in humans and to avoid an acute toxic effect resulting from shorter term infusion at faster rates.

Duration of test:

On GD 29 animals were euthanatized

No. of animals per sex per dose:

15/group

Control animals:

other: 0.9% saline at a dose volume of 21.4 mL/kg

Details on study design:

- Dose selection rationale: The dose rate was based on previous preclinical studies (unpublished). The 1 g/kg dose approximates the proposed clinical dosage. The 4.28 g/kg dose is the highest dose administered in preclinical studies that did not produce narcosis (unpublished).

- Selection of exposurre route: The intravenous route of administration was used because the lipid emulsion is intended for intravenous human administration as a component of parenteral nutrition.

- Dose volumes: 5 and 21.4 mL/kg

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (AM and PM) for mortality and moribundity. In addition, rabbits were observed once daily for clinical observations. On dosing days, animals were observed predose, immediately (within 5 min) postdose, and approximately 1 h after completion of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: daily on on GD 7 through 29; GD 0 body weights for rabbits were provided by the animal suppliers.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Feed consumption data were collected daily beginning on GD 6.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD 29

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, live fetuses were weighed, examined externally, then euthanatized
- Soft tissue examinations: Yes: all. The internal organs of the rabbit fetuses were examined in the fresh state for variations and malformations, and the sex was determined; Following completion of fetal examinations, soft tissue malformations were preserved in 10% phosphate-buffered formalin
- Skeletal examinations: Yes: all. Viscera were removed and discarded, and fetuses were processed and examined for skeletal variations and malformations; skeletal specimens were retained in glycerine
- Head examinations: Yes: all. A mid-corona. slice of the head was made to expose the internal structure of the brain for examination; the eyes were excised and examined.

Statistics:

The litter was the experimental unit for evaluation. All comparisons were made with the control group (Group 1). For rats, feed consumption, dam body weights, and fetal body weights were summarized wim means and standard deviations calculated using an Excel spreadsheet program (Microsoft Corporation). For rabbit data, Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p « 0.05, rank transformation was used to stabilize the variance. Analysis of variance [ANOVA (Winer, 1971a)] was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t test (Dunnett, 1964) was used for pairwise comparisons between groups. One-way ANOVA was used to analyze body weights, body weight changes, feed consumption, and cesarcan section data. As appropriate, rat and rabbit fetal abnormality data were analyzed by the Cochran-Armitage test (Thalcur et al, 1985) for trend and departure and by the Fisher-Irwin exact test (Thalcur et al., 1985). One-way analysis of covariance [ANCOVA (Winer, 1971b)] was used to analyze fetal body weights (males, females, and combined) with the number of fetuses in the litter as the covariate. As appropriate, for values calculated to analyze litter data or mean fetal weight data, values were first derived within the litter, and the group mean values were derived as a mean of individual litter values. Group comparisons were analyzed at the 5.0 and 1.0% two-tailed probability levels.

Indices:

early and late resorptions, % fetuses dead/live, % postimplantation loss

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: at the highest dose group

Details on maternal toxic effects:
Survival was 100% for the 1 g lipid/kg group. One animal in the control group died on GD 11 and one animal given 4.28 g lipid/kg was sacrificed after aborting on GD 20. The animal that aborted had the lowest food consumption in the group. Decreased food consumption, resulting in a decline in the health of this animal, may have contributed to the abortion. The abortion was not considered to be test article-related as it was in the range of historical control incidence. There were no remarkable necropsy findings for either animal. Clinical observations in the test article-treated groups were limited to faecal findings (i.e., increased incidence of few or no faeces). Three animals each at 4.28 g lipid/kg had no faecal output for 1 day during the dosing period.
The data on maternal body weight changes as taken from the tabellary reporting showed a significant loss of body weights during gestational day 7-20 in maternal rabbits accompanied by a reduced food consumption.
The body weight gain during GD 7-20 was found to be 95.4 g in control animals and 119.6 g in animals dosed with 1000 mg/kg bw/d, whereas at the same time animals dosed with 4280 mg/kg bw/d showed a significant loss of body weight of - 124.8 g (p<0.01).
Food consumption in the highest dose group was only 50% of the control during GD 12-13 and only 10% of the control during GD19-20. Food consumption continued to be significantly lower for the highest dose group during the early post-treatment period (GD 20 to 24), but recovery was noted at a later interval (GD 24 to 29). The decreased food consumption observed in this study was an expected occurrence based on the high-caloric nature of the test article. There were no test article-related findings at necropsy. All pregnant animals had at least one viable foetus at scheduled caesarean section on GD 29 (i.e., no dams had total resorptions).

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Remarks:

(i.v. application)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: non-adverse effects due to maternal toxicity occurred at the highest dose

Details on embryotoxic / teratogenic effects:
The mean percentage of total resorptions/ litter (postimplantation loss) was significantly higher and the mean percentage of live foetuses/litter was correspondingly lower in the 4.28 g lipid/kg group.
Mean covariate-adjusted foetal body weights (males, females, and combined) for the 4.28 g lipid/kg group were significantly lower.

The proportion of foetuses and litters in the 4.28 g lipid/ kg group with external morphological abnormalities was significantly higher than that of the control group.
The most notable findings were rachischisis and short tail seen in three and two high-dose litters, respectively. Single incidences of the following malformations were also present
in the high-dose group: exencephaly, ablepharia, exophthalmus, and ectrodactyly. Single litter incidences of carpal flexure, tarsal flexure, and malpositioned shoulders were noted for the 1 g lipid/kg group; however, these were not considered to be test article-related. No external abnormalities were noted for the control group.

The total incidence of litters in the 1 g lipid/kg group with foetuses having soft tissue abnormalities was significantly higher than that of the control group; however, no significant differences were present when individual malformations and variations were analyzed statistically. Although the incidence of litters in the 4.28 g lipid/kg group with viscerally abnormal foetuses was also higher than controls, the difference was not statistically significant. In general, soft tissue abnormalities were present in the test article-treated groups as single foetal or litter incidences restricted to three or four litters/group, suggesting a litter-related occurrence.
Because no soft tissue abnormalities (with the exception of absent azygous lobe of the lung) were noted for the control litters, the relationship of the abnormalities in the test article groups to the test article is inconclusive.
The proportions of foetuses with variations in the 4.28 g lipid/kg group [skull bones unossified, more than 26 presacral vertebrae, and 12 full pairs of ribs (litter incidence was also greater)] and total skeletal abnormalities were significantly higher than those in the control group. The proportions of litters in this group with foetuses having misaligned sternebrae, more than 12 full pairs of ribs, and fused ribs were also significantly higher than those in the control group.
Though not statistically significant, there was a notable increase in the number of high-dose foetuses/litter with malformations of the vertebral column. The anomalies were varied in location (cervical, thoracic, sacral, and caudal) and type (malformed, misaligned, absent, and fused). The incidence of any one of these findings was not markedly higher than that of the controls (usually only one litter was affected with a given abnormality); however, when combined, the incidence of foetuses/litters from the high-dose group with vertebral column malformations was notably higher than that of the control group.

Key result

Dose descriptor:

NOAEL

Effect level:

4 280 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

iv application

Basis for effect level:

other: Developmental toxicity

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

iv application

Basis for effect level:

other: Developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Cesarean section examinations of rabbits revealed higher postimplantation loss (i.e., increased incidence of resorptions) and, correspondingly, fewer live foetuses at the 4.28 g lipid/kg level. Mean foetal weights were lower for this group as well, and more litters in the group tended to have foetuses with morphological anomalies than were seen in the control group. Reduced foetal weights may have been secondary to the decreased maternal food consumption observed at this dose level. Similar findings were not present for rats. Because treated females were consuming significantly less food than control females, the foetal effects noted for rabbits (especially the postimplantation loss and decreased foetal weights) may have been due to dietary deprivation, as opposed to a direct effect by the test article. Increased incidence of abortion and implant resorption among pregnant rabbits subjected to dietary deprivation have been reported (Matsazawa et at, 1980). There were no related patterns of major malformations for foetuses. Skeletal variations were present in all litters, including control and treated groups. Diverse abnormalities were seen that may have been associated with maternal toxicity (Khera, 1984; Manson, 1986).

Administration of the test article to rabbits at 1 or 4.28 g lipid/kg resulted in a NOEL for developmental toxicity greater than or equal to 1 g lipid/kg but less than 4.28 g lipid/kg based on the foetal findings (increased postimplantation loss, lower foetal body weights, and higher incidence of morphological anomalies) seen at the 4.28 g lipid/ kg level. Administration of the test article at the highest dose also resulted in lower maternal food consumption and loss of body weight, therefore to observed foetal effects were interpreted as secondary effects of maternal toxicity and not as teratogenic effects.

 

Conclusions:

Upon intravenous infusion of 1000 mg/kg bw/d during gestational days 7-19, medium chain triglycerides exerted no teratogenic effects in rabbits.

Executive summary:

In a developmental toxicity study medium chain triglycerides (MCT) was administered to 15 females Hra:(NZW)SPF rabbits/dose by intravenous infusion at dose levels of 1000, and 4280 mg/kg bw/day from days 7 through 19 of gestation.

The maternal LOAEL is 4280 mg/kg bw/day, based on strongly reduced food consumption, significant loss of body weight, no faecal output in some animals.The maternal NOAEL is 1000 mg/kg bw/day.

The developmental NOAEL is 4280 mg/kg bw/day. Increased resorptions, decreased foetal body weights, and increased incidence of morphological anomalies are reported at 4280 mg/kg bw/d. These foetal effects were due to the result of dietary deprivation, maternal toxicity, or both, rather than a direct teratogenic effect of the test article.

The developmental toxicity study in the rabbit is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rabbits.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

parenteral route of application, only two dose levels, only two dose levels, number of corpora lutea not reported

Principles of method if other than guideline:

In this study, we evaluate the developmental toxicity of a 20% lipid emulsion that contains a 3:1 ratio of medium chain triglyceride (MCT) to one long chain containing lipid emulsion (LCT).

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: Crl:CD BR rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Portage, MI)
- Weight at study initiation: 150 to 200 g on Gestation Day (GD) 0
- Housing: individually in suspended stainless steel cages
- Diet (e.g. ad libitum): ad libitum except during dose administration
- Water (e.g. ad libitum): ad libitum except during dose administration

ENVIRONMENTAL CONDITIONS
Environmental controls in the animal rooms were set to maintain temperature, relative humidity, and light/dark cycle

Route of administration:

intravenous

Vehicle:

other: 20% lipid emulsion was created, no further specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose was administered daily to rats by intravenous infusion via a caudal vein using a Quik-Cath Teflon catheter connected to a syringe using an extension set. Doses for rats and rabbits were delivered using syringe pumps.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

Time-mated Crl:CD BR rats were shipped by the supplier on GD 4.

Duration of treatment / exposure:

GD 6 through 15

Frequency of treatment:

daily, for approximately 4 h/day
A 4-h infusion was used to approximate longer-term infusion in humans and to avoid an acute toxic effect resulting from shorter term infusion at faster rates.

Duration of test:

On GD 20 animals were euthanized

No. of animals per sex per dose:

rats: 25 or 29/group

Control animals:

other: 0.9% saline at a dose volume of 21.4 mL/kg

Details on study design:

- Dose selection rationale: The dose rate was based on previous preclinical studies (unpublished). The 1 g/kg dose approximates the proposed clinical dosage. The 4.28 g/kg dose is the highest dose administered in preclinical studies that did not produce narcosis (unpublished).

- Selection of exposure route: The intravenous route of administration was used because the lipid emulsion is intended for intravenous human administration as a component of parenteral nutrition. The dose was administered daily to rats by intravenous infusion for approximately 4 h/day on GD 6 through 15 via a caudal vein using a Quik-Cath Teflon catheter connected to a syringe using an extension set.

- Dose volumes: 5 and 21.4 mL/kg bw

Maternal examinations:

CLINICAL SIGNS AND CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed twice daily (AM and PM) for mortality and moribundity. On dosing days, animals were observed predose, immediately (within 5 min) postdose, and approximately 1 h after completion of dosing.

BODY WEIGHT: Yes
- Time schedule for examinations: daily on GD 5 through 20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: Food consumption data were collected daily beginning on the day of receipt

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day GD 20

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: half litter; live fetuses were weighed, examined externally, sexed, then euthanatized
- Soft tissue examinations: half litter; Following completion of fetal examinations, soft tissue malformations were preserved in 10% phosphate-buffered formalin
- Skeletal examinations: half litter; Viscera were removed and discarded, and fetuses were processed and examined for skeletal variations and malformations; skeletal specimens were retained in glycerine
- Head examinations: half litter; A mid-coronal slice of the head was made to expose the internal structure of the brain for examination; the eyes were excised and examined.

Statistics:

The litter was the experimental unit for evaluation. All comparisons were made with the control group (Group 1). For rats, feed consumption, dam body weights, and fetal body weights were summarized wim means and standard deviations calculated using an Excel spreadsheet program (Microsoft Corporation). For rabbit data, Levene's test (Levene, 1960) was done to test for variance homogeneity. In the case of heterogeneity of variance at p « 0.05, rank transformation was used to stabilize the variance. Analysis of variance [ANOVA (Winer, 1971a)] was done on the homogeneous or transformed data. If the ANOVA was significant, Dunnett's t test (Dunnett, 1964) was used for pairwise comparisons between groups. One-way ANOVA was used to analyze body weights, body weight changes, feed consumption, and cesarcan section data. As appropriate, rat and rabbit fetal abnormality data were analyzed by the Cochran-Armitage test (Thalcur et al, 1985) for trend and departure and by the Fisher-Irwin exact test (Thalcur et al., 1985). One-way analysis of covariance [ANCOVA (Winer, 1971b)] was used to analyze fetal body weights (males, females, and combined) with the number of fetuses in the litter as the covariate. As appropriate, for values calculated to analyze litter data or mean fetal weight data, values were first derived within the litter, and the group mean values were derived as a mean of individual litter values. Group comparisons were analyzed at the 5.0 and 1.0% two-tailed probability levels.

Indices:

early and late resorptions, % fetuses dead/live, % postimplantation loss

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The high-dose group consistently exhibited lower body weights beginning 1 day after dose administration throughout the remainder of the study. Feed consumption was also notably lower for nine of the ten days during dosing, with an increase in feed consumption after completion of dose administration (GD 15). The decrease in feed consumption at the high-dose level was expected based on the high-caloric nature of the test article.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

There was a trend toward an increasing incidence of necropsy findings in the high-dose group, including enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. These changes indicated that the high-dose group was likely exhibiting test article effects.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: effects in the highest dose group

Details on maternal toxic effects:
The only test article-related findings observed were associated with tail lesions in both test article groups and the occasional occurrence of red tinged urine (8 of 29) and vaginal bleeding (1 of 29) in the high-dose group. The tail findings were predominantly those of discoloration and ulceration. Incidences were 1/25, 14/25, and 23/29 for these tail lesions in the control, low-, and high-dose groups, respectively, and ranged from mild to severe with some necrosis and partial loss of the tail.
These findings were generally considered to be related to occurrences of observed extravasation of the MCT:LCT lipid test article into perivascular areas. Evaluation of urine collected from one high-dose animal suggested a bacterial infection of the urogenital tract. Clinical observations for this animal included occasional red-tinged urine and vaginal bleeding. This rat was noted as having a large urinary bladder stone and kidney hydronephrosis at necropsy.
There were no marked differences in mean body weights or feed consumption for the low-dose group compared with those of the control group. However, the high-dose group consistently exhibited lower body weights beginning 1 day after dose administration throughout the remainder of the study. Feed consumption was also notably lower for nine of the ten days during dosing, with an increase in feed consumption after completion of dose administration (GD 15). The decrease in feed consumption at the high-dose level was expected based on the high-caloric nature of the test article.
Necropsy findings were primarily related to tail effects and were observed for most rats in the high-dose group and some rats in the low-dose group. In addition to tail effects, there was a trend toward an increasing incidence of necropsy findings in the high-dose group, including enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. These changes indicated that the high-dose group was likely exhibiting test article effects.
There was a slight trend toward decreasing mean gravid uterine weights in proportion to increasing test article dose; however, due to the large variability between groups, group mean uterine weights appeared to be similar. With the exception of one control dam, all females were pregnant and had at least one viable fetus/litter (i.e., no dams had total resorptions).

Dose descriptor:

NOAEL

Effect level:

4 280 mg/kg bw/day

Based on:

test mat.

Remarks:

(i.v. application)

Basis for effect level:

other: developmental toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Remarks:

(i.v. application)

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no significant group differences in preimplantation or postimplantation loss or in the mean percentage of live or resorbed fetuses; no dead fetuses were present. Mean fetal sex ratios of the test article-treated groups were comparable with those of controls. There were no apparent effects on mean fetal body weight (combined, males, or females). There were no test article-related fetal external, soft tissue, or skeletal observations.
A high incidence of folded retina in control and test article-treated groups was attributed to shrinkage of the retina during storage in alcohol prior to being transferred to Bouin's fixative.
Omphalocele and cleft palate were observed in one fetus each in the control and high-dose groups, respectively. The only fetal skeletal malformation observed (malformed/misshapen skull bones) was present in one fetus each from two control litters. Fetal skeletal variations were present in control and test article-treated groups in a nondose-related pattern.

Dose descriptor:

NOAEL

Effect level:

4 280 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

iv application

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Upon intravenous infusion of 4280 mg/kg bw/d during gestational days 6-15, medium chain triglycerides exerted no teratogenic effects in rats.

Executive summary:

In a developmental toxicity study medium chain triglycerides (MCT) was administered to 25 or 29 Crl:CD BR rats/dose by intravenous infusion at dose levels of 1000, and 4280 mg/kg bw/day from days 6 through 15 of gestation.

The maternal LOAEL is 4280 mg/kg bw/day, based on an increasing incidence of necropsy findings, including enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. The maternal NOAEL is 1000 mg/kg bw/day.

No adverse effects related to prenatal development have been reported. The developmental NOAEL is 4280 mg/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the main guideline requirement for a developmental toxicity study (OECD 414) in rat.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

4 280 mg/kg bw/day

Study duration:

subacute

Species:

other: rat and rabbit

Quality of whole database:

Sufficient to address requirements.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the findings of reliable repeated dose and developmental toxicity studies conducted on structural analogues, classification of the substance is not justified.