Tall oil, sodium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1998

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:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Stigmast-5-en-3-β-ol

EC Number:

201-480-6

EC Name:

Stigmast-5-en-3-β-ol

Cas Number:

83-46-5

Molecular formula:

C29H50O

Constituent 2

Reference substance name:

(24R)-ergost-5-en-3β-ol

EC Number:

207-484-4

EC Name:

(24R)-ergost-5-en-3β-ol

Cas Number:

474-62-4

Molecular formula:

C28H48O

IUPAC Name:

(3S,8S,9S,10R,13R,14S,17R)-17-[(2R,5R)-5,6-dimethylheptan-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

Constituent 3

Reference substance name:

Stigmasta-5,22-dien-3-β-ol

EC Number:

201-482-7

EC Name:

Stigmasta-5,22-dien-3-β-ol

Cas Number:

83-48-7

Molecular formula:

C29H48O

IUPAC Name:

(3S,8S,9S,10R,13R,14S,17R)-17-[(E,2R,5S)-5-ethyl-6-methylhept-3-en-2-yl]-10,13-dimethyl-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol

Constituent 4

Reference substance name:

Linoleic acid

EC Number:

200-470-9

EC Name:

Linoleic acid

Cas Number:

60-33-3

Molecular formula:

C18H32O2

IUPAC Name:

octadeca-9,12-dienoic acid

Specific details on test material used for the study:

This study used sterol/fatty acid esters as test material, since this is the chemical form in which they are extracted from plants

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Alpk:APfSD

Details on species / strain selection:

Strain used as the test lab had substantial background data available for it.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit of Zeneca Pharmaceuticals (Alderley Park, Macclesfield, IJK)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: Approximately 145-190g
- Fasting period before study:
- Housing:
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: Up to 2 weeks

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: feed

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): The phytosterol ester was added to the diet (modified form of the AIN (American lnstitute of Nutrition) diet) to give concentrations of 0.16, 1.6,.3.2 and 8.1% (w/w) equivalent to phytosterol ester concentrations of 0.1, 1.0, 2.0 and 5.0% (w/w), respectively. The dry ingredients were mixed in a mixer for approximately 1 minute. The phytosterol ester was then mixed with the maize oil component of the diet using a blender. The oil mixture was then added to the dry ingredients and the complete diet
mixed for 6 minutes.
- Storage temperature of food: A 20-day stability trial conducted at both ambient temperature (19-25°C) and refrigerator temperature (0-4°C) showed PE to be stable in the diet. It is not clear which of these temperatures was used for storage.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the start of the study the homogeneity and stability of phytosterol ester in the experimental diets was established. The homogeneity of three dietary phytosterol ester concentrations was determined (0.1, 1.0 and 10% w/w) to cover the range of dose levels anticipated for the feeding study. Each dietary dose level was determined by taking five separate samples for analysis from the following positions in the mixing bowl: top centre, middle centre, bottom centre, left centre, right centre). The diets were analysed on the day of preparation (from the homogeneity samples) which also constitute day 0 of the stability trial.

Four additional samples were taken from each diet; two were stored at ambient temperature (19-25°C) and two were stored refrigerated (0-4°C). A pot from each storage temperature was then analysed on day 13 or day 20 to determine the concentration of phytosterol ester. In addition, the actual achieved concentration of phytosterol ester used in the feeding study was determined by analysis of all dose levels for diets prepared for use in week 1, 7 and 13. In each case the diet samples were extracted with petroleum spirit and analysed by HPLC with UV detection at 210 nm and quantitation was achieved by comparison with standards of known concentrations.

Recovery data showed the method of analysis to be acceptable for levels of phytosterol ester in the diet in the range 0.1% to 10% (w/w) and indicated an error bar of ±20% for the 0.1% level and ±15% for the 1.0% and 10% levels. Homogeneity of mixing the diets was demonstrated, within experimental error, for diet samples prepared at 0.1%, 1.0% and 10% (w/w). A 20-day stability trial conducted at both ambient temperature (19 - 25°C) and refrigerated temperature (0-4°C) showed phytosterol esters to be stable in the diet. The actual concentrations of phytosterol esters in the three batches of diet prepared at week 1, 7 and 13 were within ±12% of the nominal concentrations and within the accepted experimental error of the analytical method used.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuous in diet

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.16 other: % (w/w) phytosterol ester

Remarks:

Equivalent to phytosterol concentration of 0.1% (w/w)

Doses / concentrations 2

Dose / conc.:

1.6 other: % (w/w) phytosterol ester

Remarks:

Equivalent to phytosterol concentration of 1.0% (w/w)

Doses / concentrations 3

Dose / conc.:

3.2 other: % (w/w) phytosterol ester

Remarks:

Equivalent to phytosterol concentration of 2.0% (w/w)

Doses / concentrations 4

Dose / conc.:

8.1 other: % (w/w) phytosterol ester

Remarks:

Equivalent to phytosterol concentration of 5.0 % (w/w)
Equivalent to phytosterol dose of 4100 mg/kg bw/day and phytosterol ester dose of 6600 mg/kg bw/day

No. of animals per sex per dose:

20

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: No satellite groups
- Post-exposure recovery period in satellite groups: No post exposure group

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: On day 1 then weekly thereafter (at the same time as body weight measurements).

BODY WEIGHT: Yes
- Time schedule for examinations: On day 1 then weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Continuous

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: An ophthalmoscopic examination of all the animals was conducted prior to the start of the study. The eyes of rats from the control and high dose groups were also examined during week 13. The examination was carried out using a Fison's binocular indirect ophthalmoscope after instillation of 0.5% (w/w) tropicamide into the eyes to dilate the pupils.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the 90 day feeding period.
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: Not specified
- How many animals: All animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the 90 day feeding period.
- Animals fasted: Not specified
- How many animals: All animals
- Parameters checked in table [No.1] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 2)

HISTOPATHOLOGY: Yes (see table 2)

Statistics:

Body weights were considered by analysis of covariance on initial (week 1) body weight, separately for males and females. Weekly food consumption, food utilisation and weekly water consumption were considered by analysis of variance, separately for males and females.
Haematology and clinical chemistry were considered by analysis of variance. Male and female data were analysed together and results examined to determine whether any differences between control and treated groups were consistent between sexes.

Organ weights were considered by analysis of variance and analysis of covariance on final body w eight, separately for males and females. All data were evaluated using the GLM procedure in SAS version 6.12 (1989). Least squares means for each group were calculated using the LSMEAN option in SAS PROC GLM. Analysis of variance and covariance allowed for the replicate structure of the study design. Unbiased estimates of differences from control were provided by the difference between each treatment qroup least-squares mean and the control group least-squares mean.

Differences from the control were tested statistically by comparing each treatment group least squares mean with the control group least squares mean using a two sided Student's t-test based on the error mean square in the analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

Three rats of the low dose group were killed for humane reasons, but this was not related to the test substance.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Group mean food consumption for males receiving phytosterol ester at dietary levels of 1.6%, 3.2% and 8.1% was slightly higher than that of concurrent controls on occasions during the study. This was probably due to the lower energy content of the diets due to the phytosterol component and wasn't considered to be of any toxicological significance.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Only minor statistically significant changes observed, which were not related to the test substance.
Slightly reduced platelet counts were observed for females at all doses, in comparison with concurrent controls. However, the magnitude of these changes was not sufficient to be considered biologically significant; for example, a 50-fold increase in dietary phytosterol levels from 0.16% to 8.1%, resulted in only a 3% reduction in platelet count. While slightly reduced platelet counts were also observed for males receiving phytosterol ester at dietary levels of 1.6% and
3.2%, group mean platelet count for males receiving phytosterol ester at the highest dietary level of 8.10% was not statistically different from that of controls.

Marginally reduced prothrombin time as observed in females at all doses and marginally increased APTT time was observed for males receiving phytosterol ester at dietary levels of 3.2% and 8.1%. These differences from control values were very small and were considered to be of no biological or toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Only minor statistically significant changes observed, which were not related to the test substance.

Minor increases in plasma albumin, phosphorus, or magnesium levels and slight increases in several plasma or serum enzymes (including plasma alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, creatinine kinase and lactate dehydrogenase activities and serum 5'-nucleotidase activity) were observed for males and/or females at dietary phytosterol ester levels of 1.6%, 3.2% and 8.1%. However, in the absence of any significant organ weight changes
or histopathological findings in these animals, the changes were concluded not to be biologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant changes to organ weights in comparison with the control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no macroscopic findings that could be attributed to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no microscopic findings that could be attributed to treatment.

A slightly increased incidence of microlithiasis was observed in the kidney cortex of females of the high dose group. This is a common finding of variable incidence in the kidneys of female rats of this strain and was not considered to be an adverse effect.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

>= 8.1 other: %

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

2

Dose descriptor:

NOAEL

Effect level:

>= 6 600 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: phytosterol ester

3

Dose descriptor:

NOAEL

Effect level:

>= 4 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: phytosterol

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a 90-day repeated dose toxicity study (reliability score 1) conducted using a protocol equivalent to OECD 408 and in compliance with GLP, administration of phytosterol in the diet of Wistar rats for 90 days did not lead to any adverse effects up to dietary concentrations of 8.1% phytosterol ester (equivalent to 6600 mg/kg bw/day; or 4100 mg/kg bw/day phytosterol).