Fatty acids, coco, esters with oxybis(propanediol)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Subchronic Toxicity Studies of SALATRIM Structured Triacylglycerols in Rats. 3. Triacylglycerols Composed of Stearate, Acetate, Propionate, and Butyrate

Author:

Hayes, J.R. et al.

Year:

1994

Bibliographic source:

J. Agric. Food Chem. 42, 552-562

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

short- and long-chain triacylglycerols (SALATRIM 234CA and SALATRIM 234CS)

IUPAC Name:

short- and long-chain triacylglycerols (SALATRIM 234CA and SALATRIM 234CS)

Test material form:

liquid

Specific details on test material used for the study:

Total fatty acid profile data for the higher molecular weight groups (>=C12:0) were obtained by saponification of the triacylglycerol mixture with methanolic sodium hydroxide followed by esterification with methanolic boron trifluoride. Methyl esters of the fatty acids were quantitated by gas chromatography. For lower molecular weight groups (External standards were used, and quantitation was based upon peak height.
Free fatty acid (FFA) concentration and peroxide value (PV) analyses were conducted by NFG. FFA concentrations were determined by titration using AOCS Official Method Ca 5a-40 (AOCS, 1990a). The FFA concentrations and PVs for the two SALATRIM triacylglycerol mixtures were low. For SALATRIM 234CA lot A019, the FFA concentration and PV were 0.07 +/- 0.01 wt % and 0.33 +/- 0.05 mequiv of peroxide/kg, respectively. The results for SALATRIM 234CS lot A018 were 0.19 +/- 0.03 wt % and 0.87 +/- 0.04 mequiv of peroxide/kg, respectively.
Commercially available Mazola corn oil was used as the control fat.

Test animals

Species:

rat

Strain:

other: CrLCD'BR VAF/Plus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Portage, MI)
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: 150-201 g for males and 114-158 g for females
- Fasting period before study:
- Housing: Animal husbandry complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication 86-23,1985). Rats were identified by ear tags and housed singly in stainless steel, wire-bottom cages in an animal room
- Diet: ad libitum except when rats were fasted overnight before blood collection and necropsy.
- Water: ad libitum during all phases of the study.
- Acclimation period: 2 weeks before study initiation

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency) and storage: Test diets were prepared biweekly and divided into two portions. The first portion was fed during the first of the two weeks, and the second portion was stored frozen (-5 to -20 °C) until fed during the second week.
- Mixing appropriate amounts with (Type of food): The SALATRIM fats and corn oil were mixed with powdered NIH-07 Rat and Mouse Ration 5018 (Purina Mills, Inc.) and fed ad libitum except when rats were fasted overnight before blood collection and necropsy.

Drinking water was provided ad libitum during all phases of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diets were analyzed for content of either corn oil (by gravimetric analysis) or the SALATRIM fats (by supercritical fluid chromatography).
Homogeneity was evaluated for the 2 % and 10% SALATRIM diets and the 10% corn oil diet mixed for week 1. Stability of the SALATRIM fats in the diets for up to 15 weeks when frozen (-5 to -20°C) was assessed by analysis of samples of the 2% and 10% diet mixtures.

Duration of treatment / exposure:

Rats were fed for at least 13 weeks.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 other: % of the diet by weight

Doses / concentrations 2

Dose / conc.:

2 other: % of the diet by weight

Doses / concentrations 3

Dose / conc.:

5 other: % of the diet by weight

Doses / concentrations 4

Dose / conc.:

10 other: % of the diet by weight

No. of animals per sex per dose:

20

Control animals:

yes

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, moribundity, and signs of toxicity. For all rats except the sentinel group, physical examinations were conducted weekly.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily for mortality, moribundity, and signs of toxicity. For all rats except the sentinel group, physical examinations were conducted weekly.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment, weekly thereafter, and at necropsy.

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, individual feed consumption was measured weekly during treatment. Daily SALATRIM 234CA lot A019 consumption for the 2%, 5%, and 10% groups averaged 1.4, 3.5, and 6.9 g/kg in males and 1.7,4.0, and 7.9 g/kg in females, respectively, during the 13-week study.
Daily SALATRIM 234CS lot A018 consumption for the 2%, 5%, and 10% groups averaged 1.4, 3.4, and 6.7 g/kg in males and 1.6, 4.0, and 7.6 g/kg in females, respectively.
Daily consumption of corn oil averaged 6.1 and 7.0 g/kg in males and females, respectively.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and during week 13.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 13 weeks of treatment, with the exception of the sentinel group, for a standard battery of hematology
- Anaesthetic used for blood collection: Yes, Blood was collected from the retro-orbital plexus after ketamine anesthesia. Samples for hematology were collected with 10% EDTA anticoagulant, plasma for the prothrombin assay was prepared from blood collected with 3.8% sodium citrate anticoagulant.
- Animals fasted: Yes, fasted overnight for approximately 16 h before blood collection
- How many animals:10 rats per sex per group
- Parameters checked: Hematology variables were determined using a Coulter Counter S-Plus IV whole blood automated analyzer. Prothrombin time was measured using a Coag-A-Mate X2 coagulation analyzer. Differential leukocyte count and blood cell morphology slides were prepared using a Geometric Data Hemastainer and read manually.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 13 weeks of treatment, with the exception of the sentinel group, for a standard battery of serum
- Animals fasted: Yes, fasted overnight for approximately 16 h before blood collection
- How many animals: 10 rats per sex per group
- Parameters checked: Serum for the clinical chemistry determinations was prepared from blood collected without anticoagulant. Serum variables were determined using a Hitachi 704 random access chemistry analyzer except that low-density lipoprotein cholesterol. Globulin was calculated by subtraction of serum albumin from total protein and serum 25-hydroxy vitamin D (vitamin D) was measured by radioimmune assay using commercially available reagents from Incstar Corp. (Stillwater, MN).

URINALYSIS: Yes
- Time schedule for collection of urine: After 13 weeks of treatment, urine was collected from 10 rats per sex per group, with the exception of the sentinel group, for a standard battery of hematology, serum and urine chemistry, and urinalysis determinations.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, during the fasting period before blood sampling; for approximately 16 h.
- Parameters checked: Urine chemistry variables were determined using a Hitachi 704 random access chemistry analyzer and urinary fractional clearance of calcium, phosphorus, sodium, potassium, and chloride were calculated. Urinalysis was conducted manually and with the Ames Multistix. Serum trans-retinol (vitamin A) and alpha-tocopherol (vitamin E) concentrations were determined by highpressure liquid chromatographic methods.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, At terminal necropsy, following 13 weeks of treatment, all rats in groups 1-8 were subjected to gross necropsy. Adrenals, brain, kidneys, liver, and testes were weighed. After the liver of each rat selected for serum vitamin chemistry was weighed, the left medial lobe was removed and frozen in liquid nitrogen and stored frozen at -70°C until analyzed for vitamins A and E using high-performance liquid chromatographic methods. The entire femur not used for histopathology from each rat selected for clinical pathology was removed and stored frozen at -20°C. Defatted dry weight and percent ash of femurs were determined. Each femur was assayed for barium, calcium, copper, iron, magnesium, phosphorus, potassium, sodium, strontium, and zinc concentrations by inductively coupled plasma spectrometry.

HISTOPATHOLOGY: Yes, a complete set of tissues was collected from all rats and fixed in standard fixatives. Tissues from the control rata and rats fed 10% SALATRIM 234CA lot A019 and 10% SALATRIM 234CS lot A018 were subjected to histopathology. Macroscopic lesions (if any), lungs, liver, and kidneys from all rats (except sentinel)
were subjected to histopathology. Tissues for histopathological examination were embedded, sectioned, stained with hematoxylin and eosin, and examined by light microscopy.

Statistics:

Statistical analyses were conducted for the following: body weights, cumulative body weight gains; food consumption; serum chemistry; hematology (except red
blood cell morphology); urine pH, volume, and specific gravity; urine chemistry; serum and liver vitamin concentrations; organ weights; organ-to-body weight percentages; organ-to-brain weight ratios; and bone mineral analyses. Levene’s test was used to test for variance homogeneity. In the case of heterogeneity of variance at p <= 0.05, transformations were used to stabilize the variance (Draper and Hunter, 1969). When necessary, the following transformations were conducted in sequence until homogeneity of variance was achieved: log10, square, square root, reciprocal, angular, and rank. Analysis of variance (ANOVA) was performed on the homogeneous or transformed data. If ANOVA was significant, the Games and Howell modified Tukey-Kramer test was used for pairwise comparisons between groups. Group comparisons were evaluated at the 5% two-tailed probability level. All statistically significant differences cited are based on comparisons with the untreated control group (group 1).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related effects were noted during daily observations and weekly physical examinations.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

All rats survived to the scheduled sacrifice except for one 10% SALATRIM 234CS lot A018 male that was sacrificed during week 12 because of a fractured calvarium. This was considered to be unrelated to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean body weights and body weight gains for male rats in the groups receiving the SALATRIM fats were comparable to those of untreated control rats, while those receiving corn oil showed slight, although not statistically significant, increases in body weight gain. Body weights and body weight gains of females were generally similar, although slightly higher weights and weight gains were observed in females fed corn oil and 10% SALATRIM 234CS lot A018.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Feed consumption (g/kg per day) for the 10% corn oil males and females was significantly lower than that of controls at all intervals.
Feed consumption (g/kg per day) for the groups given SALATRIM 234CA lot A019 and SALATRIM 234CS lot A018 generally was similar to that of the control group for each sex except that significantly lower consumption was noted during weeks 8-11 for females given 10% SALATRIM 234CS lot A018. The reason for the marked increase in week 2 feed consumption of females fed 5 % SALATRIM 234CS lot A018 and the subsequent significant decrease during week 3 is unknown. It was apparently unrelated to treatment since feed consumption in this group at all other study weeks was comparable with that of the control group.
The lower feed consumption for these rats is predictable on the basis of total caloric consumption.

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Ophthalmologic findings were comparable in treated and control rats.

Description (incidence and severity):

Hematology revealed no treatment-related effects when values for rats fed SALATRIM or corn oil were compared with controls.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Viral antibody titers were negative at initiation and termination of the study.
Statistically significant lower aspartate aminotransferase levels were noted for females fed 2% and 10% 234CA lot A019 and for females fed 2 %, 5% , and 10 % 234CS lot A018. Serum aspartate aminotransferase was not affected for males in any treatment group. No other changes in serum chemistry variables, including serum lipids, were noted for rats fed the SALATRIM fats.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In this study with SALATRIM 234CA lot A019 and SALATRIM 234CS lot A018, slightly, but not statistically significantly, increased urinary phosphorus clearance was again noted in 10% SALATRIM treated rats. No other changes in urinary mineral clearance or other urinalysis variables were observed in rats fed either SALATRIM fat.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No differences in defatted femur weights or in bone concentrations of barium, calcium, copper, iron, magnesium, phosphorus, potassium, or strontium were noted between treated and control rats of either sex in this study. Mean sodium content in bone was significantly lower in females fed 2% SALATRIM 234CA lot A019, and mean zinc content in bone was significantly higher in males fed 2 % SALATRIM 234CA lot A019 and in females fed 10% of both SALATRIM fats than in controls.
In general, there were no differences between the organ weights of rats fed either of the SALATRIM fats and untreated control rats. In the 10% SALATRIM
234CS lot A018 females, body weight collected at necropsy, absolute liver weight, and liver-to-brain weight ratio were greater and brain-to-body weight ratio was lower than controls.
The organ weight differences were considered to be related to the increased terminal body weight and were not considered to be toxicologically significant because (1) these organ weight changes occurred in one sex only, (2) no effects on the brain and liver were detected during the histopathologic examination of these females, and (3) no similar organ weight or histopathologic effects were associated with SALATRIM 234CA lot A019 in this study and no such effects were noted in previous subchronic studies with three other SALATRIM fats (Hayes et al., 1994b,c).
Body weights collected at necropsy for rats of both sexes in the 10% corn oil group were significantly increased compared with those of untreated controls. Absolute liver weights and liver-to-brain weight ratios were significantly higher for 10% corn oil-fed males than for untreated controls. A few statistically significant decreases in organ weights relative to body weights of 10% corn oil-treated rats and controls also were noted. The differences in organ weights relative to body weight were considered to be reflective of the significantly increased body weights of corn oil-treated rats. The increases in liver weight noted in corn oil-treated males also may have been reflective of the increased terminal body weight but also may have been related to the mottled livers and hepatocellular vacuolation that were observed in these males.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Macroscopically, no treatment-related effects were observed in SALATRIM-treated rats. Microscopically, an increased incidence of mineralization was noted in the
kidneys of females fed 5 % and 10 % SALATRIM 234CA lot A019 when compared with the incidence in control females. A slightly higher incidence of renal mineralization also was noted for females fed 5 % and 10 % SALATRIM 234CS lot A018 compared with controls. Except for the 10% SALATRIM 234CA lot A019-treated groups, the mineralization was similar in appearance in all groups. In the 10% SALATRIM 234CA lot A019 females, the severity of this mineralization was slightly greater than in other groups.
Mottled livers and hepatocellular vacuolation were noted for males in the 10% corn oil group. No hepatic effect was observed in SALATRIM-treated rats.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Serum and liver concentrations of vitamin E in SALATRIM treated rats were comparable to those of control rats. Mean serum vitamin A was significantly higher than control in male rats fed 2 % SALATRIM 234CA lot A019 and 10% corn oil.
Mean liver vitamin A was significantly lower than controls in 10% SALATRIM 234CA lot A019 males and 10% SALATRIM 234CS lot A018 and 10% corn oil males and females.
Mean serum 25-hydroxy vitamin D concentrations were lower than control in females fed 2% and 10% SALATRIM 234CA lot A019,2 % and 10% SALATRIM 234CS lot A018, and 10% corn oil. These lower values were statistically significant for the 2 % SALATRIM 234CA lot A019 and 2% and 10% SALATRIM 234CS lot A018 groups. No differences in serum 25-hydroxy vitamin D levels were detected in males.
Prothrombin time, an indicator of vitamin K status, was unaffected by exposure to the SALATRIM fats and corn oil.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

> 6 700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

2

Key result

Dose descriptor:

NOAEL

Effect level:

> 7 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

These data indicate that SALATRIM 234CA lot A019 and SALATRIM 234CS lot A018 are well tolerated in rats when fed for as long as 13 weeks at average daily doses in males as high as 6.9 and 6.7 g/kg, respectively, and in females as high as 7.9 and 7.6 g/kg, respectively.
The data confirm the hypothesis, based on the scientific literature, structure/activity relationships, and results of testing with similar SALATRIM fats, that these fats do not produce toxicologically significant effects in rats.
From these results, the NOAELs for repeated dose toxicity are considered to be > 6700 mg/kg/day for males and > 7600 mg/kg/day for females.