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

Developmental toxicity / teratogenicity

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1997

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Medium Chain Triglycerides (MCT)

IUPAC Name:

Medium Chain Triglycerides (MCT)

Details on test material:

- Name of test material (as cited in study report): Medium Chain Triglycerides (MCT)
- Physical state: liquid
- Analytical purity: 20% lipid emulsion designed for the use in parenteral nutrition
- Composition of test material, percentage of components: a 20% lipid emulsion containing a 3:1 ratio of MCT:LCT (Long Chain Triglycerides) from soybean oil. Medium chain fatty acids are 6 to 12 carbons in length. The 20% lipid emulsion is composed primarily of 8- and 10- carbon fatty acids and a trace of 6- and 12-carbon fatty acids. The same lot of test article was used throughout the studies, and was analytically determined to be stable for the duration of both studies. Saline (0.9% NaCl for injection, USP) was used as a nonlipid control article.
- Stability under test conditions: analytically determined to be stable for the duration of both studies

Test animals

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

Administration / exposure

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

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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).

Effect levels (maternal animals)

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

Results (fetuses)

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.

Effect levels (fetuses)

1

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

2

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

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

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.

 

Applicant's summary and conclusion

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.