Tall oil, sodium salt

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

Molecular formula:

C28H50O

IUPAC Name:

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

Test material form:

other: waxy solid

Details on test material:

The purity of the test sample is approximately 99%

The test substance consisted of stanol fatty acid esters derived from wood (tall oil) or vegetable oil precursors obtained from Raision Tehaat OY AB, Raisio, Finland.

The sample identified as Sito-70 stanol ester contained 57.08% total stanols /l00g fat (68% sitostanol, 30% campestanol, 2% unsaturated sterol), 41.96% fatty acids, and, 2% unsaturated sterols and unknowns. Approximately 93.4% of the esterified groups was C-18 fatty acids, with 3.6% C-16, 2.1% C-20, and 0.9% other fatty acid esters.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Netherlands BV.
- Age at study initiation: At least 12 weeks
- Weight at study initiation: 241-248 g
- Fasting period before study: None
- Housing:
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 47-88
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light):12/12

IN-LIFE DATES: No data

Administration / exposure

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
During the acclimatization period rats were fed a defined powdered diet obtained from Special Diets Services, Witham, England. The plant stanol ester product was fed to the rats in the same diet that was supplemented with rapeseed oil to equalize the energy contribution from the fatty acids in the diets with varying amounts of the stanol esters. A total of 3.68% total fatty acids from rapeseed oil and the test substance was maintained in all control and test diets with the assumption that both the test substance-derived fatty acids and rapeseed oil provide 9 kcal/g.

- Storage temperature of food: 2-10°C

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The fortified test diets were analyzed for stability, content, and homogeneity of the test substance. The analytical procedure involved acid hydrolysis, extraction
into petroleum ether, alkaline hydrolysis of the dried ether extracts, derivatization, and quantification by GC/FID. Recovery of the stanol from fortified diets varied within a range of 96.7 to 97.9% for the three test diets. The test substance was found to be stable in the diet for at least 7 days at room temperature and for at
least 39 days refrigerated at 2-10oC. Analyses showed that the stanol ester was homogeneously distributed in the diets with a coefficient of variation of <4% for five samples of each diet. The actual versus intended content of total stanols in the three test diets varied from 91.8 to 97.5%.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 male to 2 females
- Length of cohabitation: until evidence of mating
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy:sperm in vaginal smear referred to as day 0 of pregnancy.

Duration of treatment / exposure:

Day 0 to 21 of gestation

Frequency of treatment:

Daily

Duration of test:

22 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: % total stanols

Remarks:

Equivalent to 1.75% plant stanol esters

Doses / concentrations 2

Dose / conc.:

2.5 other: % total stanols

Remarks:

Equivalent to 4.38% plant stanol esters

Doses / concentrations 3

Dose / conc.:

5 other: % total stanols

Remarks:

Equivalent to 8.76% plant stanol esters

No. of animals per sex per dose:

28 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: No data
- Rationale for animal assignment (if not random): The mated females were distributed over the control and treatment groups such that the rats that became pregnant on the same day were equally distributed over all groups. Females impregnated by the same male were placed in different treatment groups.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: From the details in the publication, the exact nature of the observations is not clear.

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 7, 14 and 21 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, food intake was measured for gestation days 0-7, 7-14 and 14-21.
- 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

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: Details of examination not provided.

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
- Other: Number of live and dead fetuses, sex of fetuses, weights of ovaries, empty uteri, fetuses and placentas.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

Statistical evaluations of the equality of means for body weights, body weight gain, organ weights, and food consumption data were performed by a one-way
analysis of variance (ANOVA) followed by Dunnett's multiple-comparison test. Clinical findings and fetopathological data were evaluated by Fisher's exact
probability test. Differences in the number of pregnant females and females with live fetuses were evaluated using Fisher's exact probability test. Numbers of corpora lutea, implantations, live and dead fetuses, and early and late resorptions were evaluated by Kruskal-Wallis nonparametric analysis followed by the Mann-
Whitney U test.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

Daily clinical observations during the gestation period did not reveal any adverse findings in the rats' appearance, general condition, or behaviour in any of the groups.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights and body weight gain of pregnant females during gestation were not significantly different from the control values throughout the study in the groups fed 1 and 2.5 % total stanols. In the group fed 5% stanols a significant reduction in mean body weight relative to controls was observed at gestation days 7 and 14 but not at day 21. Body weight gains were not significantly different from control values for any group throughout gestation except for a small but
statistically significant decrease at days 0-7 for the high-dose group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption (expressed as g/animal/day) showed no significant differences from controls. A small but statistically significant increase in food consumption
(expressed as g/kg bw/day) was observed in the mid dose group during gestation days 7-14 and in the high dose group during gestations days 7-14 and 14-21.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross examinations of the maternal organs and tissues did not reveal any significant or treatment-related differences among the stanol-ester-treated and control groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 6 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed in maternal animals.

Remarks on result:

not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

A total of 682 live fetuses from 104 litters were examined for skeletal malformations, abnormalities, and variations. In 3 fetuses of 2 litters of the low dose group, missing ribs were observed. No other skeletal malformations were observed in the control or high dose groups. No significant increases in skeletal
anomalies were observed in the stanol-ester-treated groups. Skeletal anomalies were observed in 1 fetus in the low dose group that had wavy ribs, 1 fetus that had separated sternebrae, and 1 fetus that had a few ribs with reduced size. No significant differences were found in skeletal variations in any of the treated groups and the variations noted were considered to be typical observations in rat studies.

Variations in ossification were significantly greater than controls in the high dose group that had a higher incidence of fetuses with incompletely ossified frontalis and extra caudal bodies. Significant differences were also seen in the mid dose group for increased incidence of incompletely ossified frontalis and a decreased incidence of ossification of proximal phalanges of the front legs. The low dose group had a reduced incidence of fetuses with an incompletely ossified interparietalis and incidence of five to eight proximal phalanges on the hind legs but an increased incidence of incompletely ossified 0-2 metatarsals. No statistically significant differences were noted in skeletal variations when the data were analyzed on a litter basis and the incidence of variations showed no apparent relationship with increasing dose. Because of the apparent lack of a treatment-related effect, the incidence in variations in ossification were not considered to be biologically
significant.

Visceral malformations:

no effects observed

Description (incidence and severity):

No increases in visceral malformations were observed in the fetuses of groups fed with stanol esters. The incidence of visceral anomalies showed a
statistically significant decrease in the total number of fetuses with anomalies due to a decrease in the incidence of fetuses with dilated urinary bladders in the mid- and high dose groups in comparison to the controls. There was also a dose-related decrease in the incidence of fetuses with hydroureter that was significantly lower than the control at the high dose level. Although these results showed a dose-related decrease between the concentration of rapeseed oil in the diet
and dilated urinary bladder and hydroureter, there are no reports in the literature that describe a relationship between fetal developmental abnormalities and high
concentrations of rapeseed oil. Also, the incidence of dilated urinary bladder and hydroureter in the control and low dose groups was outside the upper limits of the historical control range of the testing facility. The number and type of visceral variations seen were considered to be typical for rats and no significant differences were noted among the control and treated groups.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 6 200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Remarks on result:

not determinable due to absence of adverse toxic effects

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 2 Reproductive Performance during Gestation Days 0 -21.

 

	Dietary percentage of plant stanols (percentage stanol esters)
Parameter	0	1 (1.75)	2.5 (4.38)	5 (8.76)
Females mated	28	28	28	28
Delivered prematurely (n)	0	0	0	0
Pregnant at necropsy (n)	25	28	26	25
With live fetuses (n)	25	28	26	25
With no viable fetuses	0	0	0	0
Corpora lutea (n/rat)*	14.28 ± 0.35	14.14 ± 0.32	13.73 ± 0.34	13.36 ± 0.29
Implantation sites (n/rat)*	13.36 ± 0.37	13.11 ± 0.42	12.65 ± 0.35	12.28 ± 0.50
Preimplantation loss (%/rat)*	6.29 ± 1.62	7.57 ± 1.73	7.71 ± 1.58	8.40 ± 3.14
Live fetuses (n/rats)*	13.04 ± 0.36	12.75 ± 0.46	12.35 ± 0.36	12.12 ± 0.51
Postimplantation loss (%/rat)*	2.21 ± 0.97	2.83 ± 1.39	2.56 ± 0.80	1.30 ± 0.91
Dead fetuses (n/rat)	0	0	0	0
Resorptions total (n/rat)*	0.32 ± 0.14	0.36 ± 0.19	0.31 ± 0.09	0.16 ± 0.11
Resorptions early (n/rat)*	0.32 ± 0.14	0.32 ± 0.19	0.31 ± 0.09	0.16 ± 0.11
Resorptions late (n/rat)*	0.00	0.04 ± 0.04	0.00	0.00
Total number of fetuses (n)	326	357	321	303
Male fetuses (%)	57	47^a	49	56
Female fetuses (%)	43	53^a	51	44
Gravid uterus (g)*	76.95 ± 1.72	75.29 ± 2.05	73.40 ± 1.77	71.92 ± 2.31
Empty uterus (g)*	5.12 ± 0.16	4.91 ± 0.15	4.85 ± 0.14	4.81 ± 0.14
Ovaries (g)*	0.11 ± 0.006	0.11 ± 0.004	0.11 ± 0.004	0.11 ± 0.004
Placenta weight: all fetuses (g)*	0.54 ± 0.01	0.53 ± 0.01	0.56 ± 0.01	0.55 ± 0.01
Body weight of viable fetuses (g)*	4.29 ± 0.06	4.33 ± 0.08	4.37 ± 0.07	4.34 ± 0.09
Male fetuses (g)*	4.38 ± 0.06	4.43 ± 0.09	4.49 ± 0.07	4.45 ± 0.10
Female fetuses (g)*	4.18 ± 0.06	4.24 ± 0.08	4.26 ± 0.07	4.21 ± 0.08

* values are means ±SE

a P<0.05, Fisher's exact test

Table 3 Visceral Malformations, Anomalies and Variations

	Dietary Percentage of Plant Stanols (% stanol esters)
Parameter^a	0	1 (1.75)	2.5 (4.38)	5 (8.76)
Fetuses (litters) examined	157 (25)	170 (28)	152 (26)	146 (25)
Malformations total	4 (2)	0 (0)	0 (0)	0 (0)
Brain: hydrocephaly	1 (1)	0 (0)	0 (0)	0 (0)
Kidneys: hydronephrosis	3 (1)	0 (0)	0 (0)	0 (0)
Anomalies total	30 (13)	28 (12)	7*** (5)*	8*** (5)*
Dilated oesophagus	0 (0)	0 (0)	0 (0)	1 (1)
Dilated urinary bladder	24 (10)	24 (11)	4*** (3)*	6** (4)
Ureters, hydroureter	10 (5)	5 (2)	3 (2)	2* (2)
Variations total	26 (14)	32 (16)	23 (11)	15 (8)
Stomach distended	1 (1)	0 (0)	0 (0)	0 (0)
Stomach with haemorrhagic fluid	9 (6)	6 (4)	7 (5)	7 (5)
Kidney: increased renal pelvic cavitation	13 (8)	20 (11)	12 (7)	5 (4)
Ureters: bent	6 (5)	10 (6)	12 (7)	4 (4)

a Values are incidences in fetuses (litters)

* P<0.05, **P<0.01, ***P<0.001

Table 4 Skeletal Malformations, Anomalies and Variations

	Dietary Percentage of Plant Stanols (% stanol esters)
Parameter^a	0	1 (1.75)	2.5 (4.38)	5 (8.76)
Fetuses (litters) examined	169 (25)	187 (28)	169 (26)	157 (25)
Skeletal malformations (total)	0 (0)	3 (2)	0 (0)	0 (0)
- Ribs, missing	0 (0)	3 (2)	0 (0)	0 (0)
Skeletal anomalies (total):	1 (1)	3 (2)	0 (0)	0 (0)
- Ribs, 2 or more wavy	0 (0)	1 (1)	0 (0)	0 (0)
- Ribs, 2 or more reduced size	0 (0)	1 (1)	0 (0)	0 (0)
- Sternabrae, separated	1 (1)	1 (1)	0 (0)	0 (0)
Skeletal variations (total):	41 (18)	58 (23)	51 (21)	43 (21)
- Ribs, accessory lumbar ribs	2 (1)	9 (6)	6 (5)	7 (5)
- Sternebrae, irregular shape of one	12 (10)	17 (12)	16 (13)	18 (12)
- Sternebrae, irregular shape of 2 or more	27 (13)	37 (19)	27 (14)	19 (12)
- Sternebrae, one supernumerary	1 (1)	0 (0)	5 (1)	1 (1)

a Values are incidences in fetuses (litters)

Applicant's summary and conclusion

Conclusions:

In a prenatal developmental toxicity study conducted to OECD 414 and in compliance with GLP (reliability 1) administration of stanol fatty acid esters in the diet of Wistar rats throughout gestation days 0-21 did not cause any general adverse effects on maternal animals or developmental toxicity in maternal animals or fetuses. The NOAEL from this study was at least 6200 mg/kg bw/day (the highest dose tested).