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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication meeting basic scientific principles.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
An investigation of the general, reproductive and postnatal developmental toxicity of Betapol (TM), a human milk fat equivalent
Author:
Spurgeon, M.J. et al.
Year:
2003
Bibliographic source:
Food and Chemical Toxicology 41:1355-1366

Materials and methods

Principles of method if other than guideline:
combined repeated dose, reproductive and postnatal developmental toxicity study
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Betapol (TM)
- Physical state: waxy solid at 4°C
- Analytical purity: no data
- Composition of test material: 1,3-dioleoyl 2-palmitoyl triglyceride and 1,2-dipalmitoyl 3-oleoyl triglyceride (main components)
- Stability under test conditions: the stability and homogeneity of the test material in the diet was confirmed.

Test animals

Species:
rat
Strain:
other: Crl: CD® (SD) BR VAF/Plus strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: (P) 6 weeks; (F1) 4 weeks
- Housing: animals were housed in groups of four by sex in suspended stainless steel cages during the pre-mating period. During the mating period, one male and one female were housed in plastic breeding cages. Following mating, males were re-housed in their former metal cages, while females remained in the breeding cages for birth and rearing of their offspring.
- Diet: ad libitum (standard laboratory animal diet LAD during the first week of the acclimation period for all animals and during the main study for negative controls only; standard purified diet containing 15% palm oil (reference oil) for P animals during the second week of acclimatisation)
- Water: tap water, ad libitum
- Acclimation period: approx. 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-25
- Humidity (%): 26-60
- 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:
The test diets were prepared by mixing appropriate amounts of test material with standard purified diet (American College of Nutrition, AIN).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test material in diet was confirmed (no further details reported).
Duration of treatment / exposure:
(P) Males and females: ca. 5 months (at least 10 weeks before mating and continued until weaning of all F1 litters)
(F1) Males and females: ca. 6 months (at least 10 weeks before mating and continued until weaning of all F2 litters)
Frequency of treatment:
daily, 7 days/week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1.5, 7.5 and 15%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
750, 3750 and 7500 mg/kg bw/day
Basis:
other: (assuming a daily food consumption of 50 g/kg/bw /day according to OECD ENV/JM/MONO(2000)18)
No. of animals per sex per dose:
32
Control animals:
yes, plain diet
other: diet containing 15% palm oil (reference oil)
Details on study design:
- Dose selection rationale: in a preliminary feasibility study, 15% of the test material in diet was determined to be the maximum achievable concentration mainly due to its physical nature.
- Negative controls: two negative control groups were included in the study. One group was fed AIN diet containing 15% palm oil as reference oil. The other group received LAD 2, a commercially available breeding diet for rodents containing 2.3-4.7% fat. This control group was included in order to distinguish between differences due to high oil content in the test diets and differences specifically related to the test material.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: animals were regularly handled and examined for obvious changes or reactions to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of each generation (6 and 4 weeks of age for the P and F1/F2 generations, respectively) and weekly thereafter up to terminal sacrifice. Additionally, F1/F2 animals were weighed on Day 28 post-partum and on the day of vaginal opening or balanopreputial skinfold cleavage. All females were weighed daily during mating and until parturition and on Days 0, 7, 14 and 21 post-partum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily during the first and final two weeks of the pre-mating treatment period (P generation) and daily during Weeks 5, 6, 15 and 16 of the pre-mating treatment period of the F1 generation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during the penultimate week of the pre-mating treatment period and at sacrifice of each generation
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 8 males and females of each group
- Parameters examined: packed cell volume (PCV), haemoglobin (Hb), red cell count (RBC), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), total white cell count (WBC), differential WBC counts of neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B) and monocytes, platelet counts (Plts), thrombotest (TT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during the penultimate week of the pre-mating treatment period and at sacrifice of each generation
- How many animals: 8 males and females of each group
- Parameters examined: total protein, albumin (Alb), globulin (Glob), urea nitrogen (Urea NItr), creatinine, sodium (Na), potassium (K), calcium (Ca), inorganic phosphorus (P), chloride (Cl), cholesterol (Chol), alkaline phosphatase (AP), glucose, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), non-esterified fatty acids (NEFA), phospholipid (PhLipid), total lipids (Lipid), triglycerides (Tri-glyc) and total esterified fatty acids (TEFA)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. From selected F1 and F2 weanlings, the following organs were weighed: adrenals, brain, epididymides, heart, kidneys, liver, lungs, ovaries, testes (with epididymides) and thymus (if present).
HISTOPATHOLOGY: Yes. A wide range of 51 tissues (not specified) were preserved against the contingency of subsequent microscopic examination. Selected target tissues for routine examination were adrenals, epididymides (longitudinal section), liver, pancreas, pituitary, ovaries, prostate, seminal vesicles (with coagulating gland), testes, uterus (with cervix) and vagina. From F2 females, sacrificed following sexual maturation, only tissues showing macroscopic abnormalities were preserved.
Statistics:
The primary line of comparison was within the four test groups fed diets containing 15% oil, which included the group receiving 15% reference oil and 0% test material (comparative control group).
- Bodyweight, haematology, blood chemistry and organ weights: differences between groups were examined by means of a standard statistical package based on parametric methods of central tendency such as analysis of variance and a test for trend. A test for heterogeneity was included and prompted default to non-parametric tests followed by Shirley's test, if it was significant. Extreme kurtosis in the form of 75% of values being identical prompted default to the categorical Fisher’s exact test.
- Organ weights and assessment of early post-natal development: analysis of co-variance was done using body weight as the presumptive independent variable.

The secondary line of comparison was between the group fed LAD 2 (negative or laboratory control) and the comparative control group receiving 15% reference oil and 0% test material, which thus served as a surrogate for all oil based groups. The method equated to a Student's t-test or the non-parametric equivalent.
The individual animal or mating pair was used as the experimental unit for most parameters. Weekly food and water consumption were analysed on a cage basis. For some pre-weaning values such as pup weight, the basic sample unit was the litter average.
Statistically significant differences from the comparative control group were identified using a limiting probability of p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
reference oil control and test groups: pale faecal pellets observed, presumably related to high fat content in diet (not observed in LAD group)
Mortality:
mortality observed, treatment-related
Description (incidence):
reference oil control and test groups: pale faecal pellets observed, presumably related to high fat content in diet (not observed in LAD group)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
negative (LAD) control group (f): significantly higher body weight compared with reference oil control on pregnancy Day 20 (P) and lactation Day 14 (P/F1); only marginal differences between test groups and reference oil control
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
LAD control group (P/F1): higher food consumption compared to test groups and reference oil control group
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
LAD control group (P/F1): higher water consumption compared to test groups and reference oil control group
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test groups and reference oil control group (P/F1): shorter clotting time than in LAD control group (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
test groups (F1, males ): statistically significantly higher triglyceride values after weaning compared to both controls (LAD or reference oil control)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
test groups (P/F1, f): dose-related increase in absolute ovary weight compared with reference oil control; negative (LAD) control (F1, f): significantly higher absolute weight of adrenals, but no differences between test groups and reference oil control
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
all groups (P/F1, m): liver findings included pale, mottled, enlarged, swollen, accentuated lobular markings and, most frequent of all, pale sub-capsular areas; (P/F1, f): pale sub-capsular areas
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
negative (LAD) control (P/F1, m): significantly lower incidence of liver periportal fat deposition and vacuolation; test groups (P, f): dose-dependent increase in incidence of fat deposition and vacuolation in liver
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality occurred in the groups receiving the test material or LAD over the P and F1 generations (see Table 1 under “Any other information on results incl. tables”). There was an association with blood sampling or bleeding usually after mating and rearing of litters, but no statistically significant differences in mortality. None of the deaths were considered to be treatment-related. Pale faecal pellets were consistently observed in the reference oil control and test groups. This was presumably related to the high fat content of the diet. No clinical signs were reported for the negative (LAD) control group.

BODY WEIGHT AND WEIGHT GAIN
There were only marginal differences in body weight between the reference oil control and test groups over the P and F1 generations (see Table 2 under “Any other information on results incl. tables”). In the negative (LAD) control group, body weight was consistently higher than in the reference oil control group, being statistically significantly different in females on pregnancy Day 20 (P) and lactation Day 14 (P and F1). Likewise, food and water consumption were consistently and often significantly higher in the negative (LAD) control groups (P and F1) than in the other groups, within which differences were marginal.

FOOD CONSUMPTION
Food consumption was consistently and often significantly higher in LAD negative control group compared to the other groups for both generations (P/F1) (see Table 2 under “Any other information on results incl. tables”).

WATER CONSUMPTION
Water consumption was consistently and often significantly higher in LAD negative control group compared to the other groups for both generations (P/F1) (see Table 2 under “Any other information on results incl. tables”).

HAEMATOLOGY
The clotting time in the test groups and reference oil control group of both generations (P/F1) was shorter than in the LAD control group. White blood cell counts in blood samples taken from all animals prior to mating tended to be higher than those taken after weaning of offspring of both generations. However, this effect was not treatment-related, since it both occurred in the test and control groups.

CLINICAL CHEMISTRY
Statistically significantly higher triglyceride values were found after weaning in treated males of the F1 generation compared to both controls (LAD or reference oil control). However, these effects were not observed in females, in F0 males or F1 males before weaning. Furthermore, total lipids were not elevated and no dose-response relationship was indicated. Since no concomitant change in other clinical chemistry parameters, organ weights and histopathology was noted, the increase in triglycerides was not considered to be toxicologically relevant.

ORGAN WEIGHTS
The weights of most organs were reported to be comparable and consistent across groups and generations (see Table 3 under “Any other information on results incl. tables”). The most consistent difference from the reference oil and negative (LAD) control groups was a higher absolute ovary weight in the group receiving the test material at 15% in diet. Absolute adrenal weights of the negative (LAD) control group were statistically significantly higher in females of the F1 generation and consistently but not significantly higher than those of the other groups (P and F1 generations, males and females) as stated by the authors.

GROSS PATHOLOGY
The liver was the only organ with findings of sufficient prevalence to allow intergroup comparisons (see Table 4 under “Any other information on results incl. tables”). In P and F1 males, macroscopic findings included pale, mottled, enlarged, swollen, accentuated lobular markings and, most frequent of all, pale sub-capsular areas (almost always in the median cleft). In P and F1 females, the latter was the only relevant macroscopic finding. The incidence of liver changes was statistically significantly higher in P males fed the test material at 7.5% in diet. However, there was no dose relationship in any group and convincing differences between groups were not evident.

HISTOPATHOLOGY: NON-NEOPLASTIC
A significantly lower incidence of liver periportal fat deposition and vacuolation was observed in males fed the negative control diet (LAD) (see Table 4 under “Any other information on results incl. tables”). Differences within the other groups were not conclusive. In P females fed AIN diet containing the test material, there was an apparent dose-related increase in the incidence of fat deposition and vacuolation. This effect was, however, not consistently repeated for the F1 generation. The authors suggested that the slight increases in hepatocyte vacuolisation and periportal fat deposition in treated animals were due to a physiological change probably caused by the increased fat absorption.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
P and F1 animals
Effect level:
>= 15 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Remarks:
P and F1 animals
Effect level:
>= 7 500 other: mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: assuming a daily food consumption of 50 g/kg/bw /day

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Mortality

P Generation

F1 Generation

Test material (%)

0

1.5

7.5

15.0

LAD1

0

1.5

7.5

15.0

LAD

Males started

32

32

32

32

32

28

28

28

28

28

Males dead

-

-

1

-

-

2

3

2

1

-

Associated with bleeding2

-

-

-

-

-

1

3

-

-

-

Females started

32

32

32

32

32

28

28

28

28

28

Females dead

1

-

1

-

1

1

-

-

-

-

Associated with bleeding

1

-

1

-

-

-

-

-

-

-

1Commercially available breeding diet for rodents, negative control

2Association with blood sample or bleeding usually after mating and rearing of litters

Table 2. Differences in body weight, food and water consumption (percentage difference from the reference oil control group, corresponding to 0% test material in diet)

P Generation

F1 Generation

Test material (%)

0

1.5

7.5

15.0

LAD

0

1.5

7.5

15.0

LAD

Body weight% difference

Males

0

3

3

6

5

0

-4

4

3

4

Females

0

-3

5

0

6

0

2

6

5

4

Pregnancy Day 20

0

-1

-1

-2

5*

0

1

4

3

5

Lactation Day 4

0

1

2

-1

9*

0

1

5

3

11*

Food consumption% difference

Males

0

1

0

2

28*

0

0

2

2

28*

Females

0

0

2

1

34*

0

3

1

9*

25*

Pregnancy

0

1

-4

-1

23*

0

4

8

0

5

Lactation

0

-3

4

-6

22*

0

1

1

-3

18*

Water consumption% difference

Males

0

7

5

2

31*

0

2

8

4

39*

Females

0

-2

-5

-6

15*

0

2

7

6

25*

* Statistically significantly different (p < 0.05) from the reference oil control group.

Table 3. Organ weights (P and F1 adults, F1 and F2 weanlings)

P Generation

F1 Generation

Test material (%)

0

1.5

7.5

15.0

LAD

0

1.5

7.5

15.0

LAD

Adults

No. of females

31

32

31

32

31

27

28

28

28

28

Ovaries (mg)

81.8

85.0

85.5

92.4*

88.0

85.4

92.7

93.9

99.2*

90.7

Adrenals (mg)

62.9

63.0

64.0

66.1

71.1

68.7

69.0

66.8

65.7

75.3*

F1 Generation

F2 Generation

Weanlings

No. of females

29

29

27

28

27

17

24

22

23

20

Thymus (g)

0.21

0.21

0.22

0.20

0.26

0.24

0.20

0.21

0.21

0.26

Heart (g)

0.40

0.40

0.41

0.40

0.37*

0.38

0.38

0.39

0.40

0.36

Lungs (g)

0.56

0.60

0.59

0.53

0.70

0.57

0.56

0.56

0.62

0.74*

Kidneys (g)

0.80

0.79

0.82

0.81

0.88*

0.80

0.77

0.79

0.82

0.87

Ovaries (mg)

15.8

16.4

17.7

17.8

18.4

16.6

15.4

16.3

17.9

18.3

Adrenals (mg)

18.4

19.0

18.7

17.9

20.0

16.4

16.8

16.8

17.9

20.1

* Statistically significantly different (p < 0.05) from the reference oil control group.

Table 4. Selected macro- and microscopic changes in the liver

P Generation

F1 Generation

Test material (%)

0

1.5

7.5

15.0

LAD

0

1.5

7.5

15.0

LAD

Macroscopic findings

No. of males examined

32

32

31

32

32

26

25

26

27

28

Any selected change

12

17

23*

9

11

9

9

9

13

8

Pale sub-capsular area(s)

7

9

16*

4

5

4

3

5

5

4

No. of females examined

31

32

31

32

31

27

28

28

28

28

Any selected change

9

7

5

12

6

11

6

18

12

9

Pale sub-capsular area(s)

9

7

5

12

6

11

6

18

12

9

Microscopic findings

No. of males examined

32

ne

ne

32

32

28

28

28

28

28

With vacuolation

26

ne

ne

27

6*

13

13

18

21

4*

With fat deposition

28

ne

ne

27

16*

25

25

27

26

14*

No. of females examined

32

32

32

32

32

28

28

28

28

28

With vacuolation

17

13

22

28*

15

22

10*

21

22

3*

With fat deposition

20

24

32*

30*

16

21

17*

20

25*

20

* Statistically significantly different (p < 0.05) from the reference oil control group.

ne: not examined

Applicant's summary and conclusion