Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study and GLP

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
arachidonic acid-rich oil
IUPAC Name:
arachidonic acid-rich oil
Details on test material:
The test substance is RAO, a refined arachidonic acid-rich trigylceride oil (Chemical name: 5,8,11,14-eicosatetraenoic acid-rich oil).
Composition of RAO:
Myristic acid (C14:0) = 0.28-0.41 %
Palmitic acid (C16:0) = 5.87-7.45 %
Palmitoleic acid (C16:1n – 7) = 0.19-0.29 %
Stearic acid (C18:0) = 5.26-5.89 %
Oleic acid (C18:1n – 9) = 5.48-9.27 %
Linoleic acid (C18:2n – 6) = 4.69-9.81 %
γ-Linolenic (C18:3n – 6) = 2.16-2.45 %
Arachidic acid (C20:0) = 0.80-0.89 %
Eicosenoic acid (C20:1n - 9) = 0.24-0.45 %
Eicosadienoic acid (C20:2n – 6) = 0.41-0.51 %
Eicosatrienoic acid (C20:3n – 6) = 3.54-4.06 %
Arachidonic acid (C20:4n – 6) = 41.81-43.66 %
Eicosapentaenoic acid (C20:5n – 3) = 0.08-0.40 %
Behenic acid (C22:0) = 3.18-3.42 %
Lignoceric (C24:0) = 9.41-11.98 %
Hexacosanoic (C26:0) = 1.11-1.72 %

Test animals

Species:
rat
Strain:
other: Wistar Outbred
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: F0 males ca 10 weeks, F0 females ca 11 weeks
- Housing: the rats were housed under conventional conditions, in macrolon cages. During the pre-mating period, the parent rats were housed maximally four per cage, separated by sex. During the mating period, 2 females were caged with 1 male from the same group until they mated. During gestation and lactation, the dams were housed individually. In the period between weaning and the start of the subchronic study, the F1 rats were housed one to five per cage until each cage contained five males or five females per group. In the 90-day feeding study, same sex F1 rats were housed in groups of five.
- Diet: ad libitum (commercial rodent diet RM3)
- Water: ad libitum
In all study phases, feed and water consumption was measured.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: RM3 diet
Details on oral exposure:
The study included five exposure groups composed of two control groups (a low-fat reference control group fed the RM3 standard diet and a high-fat control group fed the RM3 diet supplemented with 5 % corn oil) and three test groups that consumed diets containing 0.5%, 1.5% and 5% (wt/wt) RAO. The 0.5% and 1.5% test diets were adjussted with corn oil to 5% added fat.

Fresh batches of the test and control diets were prepared ca. once every month, sealed in airtight plastic containers under a nitrogen blanket to avoid oxidation, and stored in a freezer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The constituents and homogeneous distribution of the test substances in the diet, and the stability in the animal room (4days) or freezer (6 weeks) were confirmed by quantitative determination using GC with flame ionization detection after chloroform/methanol extraction, saponification and derivatization.
Duration of treatment / exposure:
The study was with the design consisting of two phases:
1. an in utero exposure phase in which F0 parents were fed the test or control diets starting four-weeks prior to mating, throughout mating, gestation and lactation until weaning of the F1 rats
2. a traditional 90-day feeding study in which selected F1 offspring consumed the same diets as did the F0 generation rats.
Frequency of treatment:
The diet were provided ad libitum.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5%
Basis:
nominal in diet
No. of animals per sex per dose:
1. in utero exposure phase with F0 parents: 16 females per exposure group and 8 males per exposure group
2. 90-day feeding study: F1 offspring rats 10 rats/sex/group
Control animals:
yes
Details on study design:
1. in utero exposure phase with F0 parents: 16 females and 8 males were allocated randomly to each exposure group of F0 rats, numbers that were deemed sufficient to result in at least 10 litters per group. After a pre-mating period of four-weeks, in which the F0 rats were fed their respective test or control diets, the females were mated. Vaginal smears were made daily and the day on which sperm was observed in the smears was considered gestation Day 0. The administration of test or control diets was continued throughout gestation and lactation. The morning after birth was considered post-partum Day 1. On post-partum Day 4, litters of more than eight pups were adjusted by randomly eliminating surplus pups to obtain four males and four females per litter. At post-partum Day 21, the F1 litters were weaned. All surviving female parent rats were sacrificed under CO2/O2 anesthesia shortly after pup weaning; F0 males were sacrificed and discarded without necroscopy shortly after mating.
2. 90-day feeding study: At weaning, F1 rats were randomly selected from the F0 litters (10 rats/sex/group, each selected from a different litter). during the period between weaning and the start of the 90-day feeding study, feeding of the respective test or control diets to the selected F1 rats was continued.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
1. in utero exposure phase with F0 parents

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at baseline and once every week thereafter.
Mated feamles were weighed on gestation Day 0, 7, 14 and 21. Females that gave birth were weighed on post-partum Days 1, 4, 12 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- During the pre-mating period, feed consumption of both males and females was measured per cage once per week. Feed consumption by females was recorded during pregnancy at the end of successive periods (Days 0-7, 7-14 and 14-21), and during lactation, at the end of Days 1-4, 4-7 and 7-14; feed consumption was not measured during mating.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No

REPRODUCTIVE DATA AND INDICES:
For each litter, the litter size (dead and live pups), number of male and female pups, and number of pups with external abnormalities were determined on post-partum Days 1, 4, 7, 14 and 21. the litters were weighed on post-partum Days 1, 4 (before and after culling), 7 and 14. at post-partum Day 21 all pups were weighed individually.

2. 90-day feeding study:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: at initiation of the 90-day feeding study and once per week thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- The quantity of feed consumed was measured per cage, over successive periods of 3 or 4 days.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: was measured during 5-day periods in study weeks 1, 6 and 12.

HAEMATOLOGY:Yes
Blood was collected from the abdominal aorta of all rats at necroscopy. the rats were fasted overnight but water was freely available.

CLINICAL CHEMISTRY: Yes
Blood was collected from the abdominal aorta of all rats at necroscopy. the rats were fasted overnight but water was freely available.

URINALYSIS: Yes
On Days 86-87 of the study, rats were deprived of water for 24 h. during the final 16 of those 24 h, rats were also deprived of food while urine was collected from stainless steel metabolism cages (on rat per cage) and analyzed.

Sacrifice and pathology:
Overnight-fasted rats (with access to water) were killed under CO2/=2 anesthesia on days 92-93 (males) and days 93-94 (females) and subjected to an immediate complete macroscopic examination with organ weights measured as soon as possible after dissection to avoid drying.
Following organs were weighted:
Males: Adrenals, Brain, Heart, Kidneys, Liver, Spleen, Thymus, Thyroid, Pituitary, Testes, Epididymides, Prostate
Females: Adrenals, Brain, Heart, Kidneys, Liver, Spleen, Thymus, Thyroid, Pituitary, Ovaries, Uterus
Histopathological examinations were performed on all specimens collected from rats in the high-fat control group and the 5% RAO test group.
Kidneys, liver and gross lesions from all rats in all exposure groups were also examined microscopically.
Statistics:
Means +/- standard error of the mean (SEM) were calculated. Two separate statistical analyses were conducted: the test groups were first compared with the high-fat control group, and then all groups were compared with the low-fat control group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
1 low-dose female was killed in moribund conditions on Day 30 for reasons unrelated to treatment
Mortality:
no mortality observed
Description (incidence):
1 low-dose female was killed in moribund conditions on Day 30 for reasons unrelated to treatment
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed intake tended to be lower in all high-fat groups than in low-fat controls. This effect is ascribed to the higher caloric density of the high-fat diets.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Compared to the low-fat control group, water consumption tended to be decreased in the high-fat groups in females.
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Description (incidence and severity):
see results below
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
see results below
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see results below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Intake of the test substance: Except during lactation when intake by dams doubled,, the overall RAO intakes by the F0 and F1 rats were ca. 0.3, 0.9 and 3 g/kg bwt/day in the low-, mid- and high-dose group.

Results obtained in F0 rats and observations in pups:
- clinical observations: no treatment-related clinical signs were observed
- no treatment-related effects on body weights
- no significant differences in food intake betweeen the treatment groups and the high-fat group
- feed intake in the low-fat control group was sstatistically significantly higher (12%) than in all high-fat groups during the pre-mating and the gestation period.
- no treatment-related differences in fertility and reproductive performance among the groups
- Indices for mating, female fecundity, female fertility, male fertility, gestation, birth and viability, as well as precoital and gestation times were similar among the groups.
- no relevant differences among the groups in general condition and body weights of the pups, or in viability, sex ratio or number of pubs per litter.

Results obtained in F1 rats in the subchronic study:
- the overall intake of RAO in the F1-animals was ca. 0.3, 0.9 and 3.1 g/kg bwt/day in males and 0.3, 0.9 and 3.2 g/kg bwt/day in females of the low-,mid- and high-dose group.
- clinical observations: no treatment-related clinical signs were observed, 1 low-dose female was killed in moribund conditions on Day 30 for reasons unrelated to treatment
- no treatment-related effects on body weights
- no significant differences in food intake betweeen the treatment groups and the low-fat group or the high-fat group. Feed intake tended to be lower in all high-fat groups than in low-fat controls. This effect is ascribed to the higher caloric density of the high-fat diets.

Hematology:
The statistically significant increase in monocytes noted in the high-dose group may have been treatment related, but no effect on monocytes was seen in the mid-dose group.

Clinical chemistry:
cholesterol and phospholipids (both sexs) and triglycerides (males) were decreased in high-dose rats compared to both control groups, while phospholipids were also decreased in mid-dose females. these plasma lipid decrease were expected and are ascribed to the lipid lowering effects of polyunsaturated fatty acids. Increased plasma urea and inorganic phosphate concentrations were observed in both sexes of the high-dose group; however, they differed significantly only fromm the high-fat controls (except for urea in high-dose males, which differed from both control groups). the changes may have been treatment-related but in the absence of confirmatory histopathology, the toxicological significance of these and other changes in clinical chemistry variables in the high-dose group (e.g. increased alkaline phosphatasse activity in males, and decreased aspartate aminotransferase activity in females) is doubtful. It can be concluded that there were no toxicologically significant adverse effects on clinical chemistry parameters.

Organ weights:
4 statistically significant increases were seen in absolute organ weights compared to high-fat controls: 1 in mid-dose males (liver) and 3 in high-dose females (adrenals, liver and spleen). The single sex statistically significant adrenal weight increase was not accompanied by relevant histopathological findings. Similarly, the higher relative weights of the liver and spleen were not associated with treatment-related histopathological lesions in these organs.

Effect levels

Dose descriptor:
NOAEL
Effect level:
3 170 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the findings of these 90-day deeding study, no adverse treatment-related effects for RAO were seen at up to 5% in the diet, equivalent to an overall average RAO intake of 3170 mg/kg bwt/day.
Executive summary:

In the present study according to OECD guideline 408, RAO was examined via a 90 -day feeding study with an in utero exposure.

Parent F0 animals were fed test- or control diets over 11 -12 weeks, starting four-weeks prior to mating, throughout mating, gestation and lactation until weaning of the F1 offspring. The 90 -day feeding study followed in the F1 rats.

The study included five exposure groups composed of two control groups (a low-fat reference control group fed the RM3 standard diet and a high-fat control group fed the RM3 diet supplemented with 5 % corn oil) and three test groups that consumed diets containing 0.5%, 1.5% and 5% (wt/wt) RAO. The 0.5% and 1.5% test diets were adjussted with corn oil to 5% added fat.

The administration of RAO at dietary levels up to 5% was well tolerated and affected neither health nor growth of the F0 rats, their viability, condition, body weights or pup sex ratios. Due to the higehr caloric density of the high-fat diests, feed intake in all high-fat groups of the F0 generation, including the high-fat controls, tended to be lower than in low-fat controls. Similary, there were no treat ment related reproductive or developmental index observations.

In the 90-day feeding study, F1 rats showed no treatment-related changes in general condition, neurobehavioral observations, ophthalmology findings, growth, urinalysis or histopathology. As in F0 rats, feed intake by F1 rats tended to be lower in high-fat groups.

Although statistical significance was shown for several parameters, none were considered attributable to treatment because they were (1) within historical control ranges, (2) did not demonstrate a dose-response relationship, (3) were seen with comparison to only one control, and/or (4) were seen in only one gender.

The only hematological effect not encompassed by this reasoning was increased monocyre numbers in both high-dose males and females. The statistically significant increase in monocytes noted in the high-dose group may have been treatment related, but no effect on monocytes was seen in the mid-dose group.

Among clinical chemistry parameters, cholesterol and phospholipids (both sexs) and triglycerides (males) were decreased in high-dose rats compared to both control groups, while phospholipids were also decreased in mid-dose females. these plasma lipid decrease were expected and are ascribed to the lipid lowering effects of polyunsaturated fatty acids. Increased plasma urea and inorganic phosphate concentrations were observed in both sexes of the high-dose group; however, they differed significantly only fromm the high-fat controls (except for urea in high-dose males, which differed from both control groups). the changes may have been treatment-related but in the absence of confirmatory histopathology, the toxicological significance of these and other changes in clinical chemistry variables in the high-dose group (e.g. increased alkaline phosphatasse activity in males, and decreased aspartate aminotransferase activity in females) is doubtful. It can be concluded that there were no toxicologically significant adverse effects on clinical chemistry parameters.

Organ weights: 4 statistically significant increases were seen in absolute organ weights compared to high-fat controls: 1 in mid-dose males (liver) and 3 in high-dose females (adrenals, liver and spleen). The single sex statistically significant adrenal weight increase was not accompanied by relevant histopathological findings. Similarly, the higher relative weights of the liver and spleen were not associated with treatment-related histopathological lesions in these organs.

There were no toxicologically significant adverse treatment-related effects noted for RAO at up to 5% in the diet for both males and females, equivalent to an overall average RAO intake of 3170 mg/kg bwt/day.