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Diss Factsheets

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
yes
Remarks:
Additional obs of oestrous cycling, sexual maturation & histo examinations. No total cauda epididymal sperm no., % progressively mobile sperm, % progressively motile sperm, % morphologically normal sperm & % of sperm with each identified abnormality
GLP compliance:
yes
Limit test:
no

Test material

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
Chemical structure
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

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany.
- Females, nulliparous and non-pregnant: yes
- Age at study initiation: (P) 4-5 x weeks (on receipt); (F1) x weeks
- Weight at study initiation: (P) Males: approx. 210 g; Females: approx. 125 g; (F1) Males: approx. 125 g; Females: approx. 112.5 g
- Fasting period before study: No data
- Housing: suspended stainless-steel group cages each with a wire mesh floor and front.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25
- Humidity (%): 40-70
- 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
- Rate of preparation of diet (frequency): At least every four weeks.
- Mixing appropriate amounts with (Type of food): Commercial rodent diet.
- Storage temperature of food: 2-10°C
Details on mating procedure:
At the end of the 10 week premating period:

- M/F ratio per cage: 1:1
- Length of cohabitation: three weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy.
- The mated F0 females were housed individually in suspended wire mesh cages with wire mesh floor and front.
- mated males were returned to their group cages (four animals per cage).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Several batches of diet were analysed to study homogeneity, stability and content of phytosterol ester.

Phytosterol ester was extracted from the diet with petroleum ether. The concentration of phytosterol ester in thc extracts was determined using HPLC with diode array detection (DAD) at 210 nm. Quantification of phytosterol ester was obtained by comparing the areas of the phytosterol peaks in the samples with those of calibration solutions containing known amounts of phytosterol ester.
Duration of treatment / exposure:
Approximately 32 weeks of treatment
Frequency of treatment:
Continuous in diet
Details on study schedule:
- F1 parental animals not mated until 12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: at least 15 weeks.

The same procedure was used for the F1 to F2 generation.
Doses / concentrationsopen allclose all
Dose / conc.:
1.6 other: % (w/w) phytosterol ester
Remarks:
Equivalent to a concentration of 1.0% (w/w) phytosterol
Dose / conc.:
3.2 other: % (w/w) phytosterol ester
Remarks:
Equivalent to a concentration of 2.0% (w/w) phytosterol
Dose / conc.:
8.1 other: % (w/w) phytosterol ester
Remarks:
Equivalent to a concentration of 5.0% (w/w) phytosterol.
Equivalent to 2500-9100 mg/kg bw/day phytosterol ester.
Equivalent to 1540-5620 mg/kg bw/day phytosterol.
No. of animals per sex per dose:
28
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose used was the same as previously conducted 90-day dietary study.
- Rationale for animal assignment (if not random): Random

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly for all animals in the premating period; males weekly thereafter; females on gestation days 0, 7, 14 and 21 and lactation days 1, 7, 14 and 21.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
Approximately one week after weaning of their pups, vaginal smears were made from the F0 females for 14 consecutive days. In the F1 animals vaginal opening was scored on post natal day 39.
Sperm parameters (parental animals):
Parameters examined in F0 and F1 male parental generations: testis weight, epididymis weight, prostate weight, seminal vesicles weight, preputial separation was scored in F1 animals on post natal day 31.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 ] offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: none

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: none
Postmortem examinations (parental animals):
SACRIFICE
At or shortly after weaning 10 male and 10 female F0 and F1 pups per group were subjected to a thorough necropsy. The following tissues were preserved for microscopic examination: ovaries, uterus, vagina, testes, epididymides, seminal vesicles (with coagulating glands and their fluids), prostate, pituitary, adrenals, liver and organs or tissues showing macroscopic abnormalities.

At termination all surviving parent animals of the F0 and F1 generations were killed and the following organs and tissues preserved for microscopic examination: adrenals.
brain, epididymides, liver, ovaries, pituitary, prostate, seminal vesicles with coagulating glands, testes, uterus with cervix (after counting implantation sites), vagina and all gross lesions.
Postmortem examinations (offspring):
On post natal day 21, the F1 pups were weaned and shortly after 28 males and 28 females were selected at random from as many litters as possible in each group to produce the next generation - these animals were approximately 5 weeks old at the beginning of the F1 premating period. Mating of siblings was avoided. From the remaining F1 pups, 10 male and 10 female pups were necropsied thoroughly. The remaining pups were sacrificed after an external examination only. The same procedure was used for the F2 generation.
Statistics:
Body weight, food consumption and food efficiency data were subjected to one way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test. Fisher's exact probability test was used to evaluate the numbers of mated and pregnant females, females with liveborn pups, females surviving delivery, females with (all) stillborn pups. live born and stillborn pups, pups lost at various stages, pups surviving 21 days, and male pups on postnatal days 1 and 21. Duration of gestation, litter size, and number of implantation sites per litter were evaluated by Kruskal Wallis non-parametric analysis of variance followed by Mann-Whitney U test. For evaluation of the incidence of pathological changes and clinical signs, Fisher's exact probability test was used. The time of achievement of preputial separation and vaginal opening was analysed with ANOVA followed by Dunnett's multiple comparison test. The chi -square test was used to evaluate the length of oestrus.

Reproductive indices:
For the assessment of fertility and reproductive performance the mating, fertility, fecundity, gestation and live births indices were calculated, and the sex ratio of pups was determined.
Offspring viability indices:
Viability and lactation indices were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No further details available.
Mortality:
no mortality observed
Description (incidence):
No further details available.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males: decreases were observed in weeks 2-7 and on occasions in weeks 5-15 and 18-19 in the high dose group.
Females: no statistically significant differences.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were inconsistent differences (increases and decreases) between control and treated groups.
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:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related observations (no further details available).
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester, or 1540-5620 mg/kg bw/day phytosterol
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
No further details available.
Mortality:
no mortality observed
Description (incidence):
No further details available.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Males: decreases were observed in weeks 2-7 and on occasions in weeks 5-15 and 18-19 in the high dose group, and decreases in weeks 6-9 and 11-14 of the low dose group. These changes were statistically significant.
Females: no statistically significant differences, except for day 7 of lactation in the high dose F1 generation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were inconsistent differences (increases and decreases) between control and treated groups.
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:
effects observed, non-treatment-related
Description (incidence and severity):
In males there was a small but statistically significant increase in the relative brain weight in low and mid dose groups only.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No further details available.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No further details available.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester or 1540-5620 phytosterol
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No further details available.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pup mortality was increased on post natal day 4. Although the differences reached statistical significance, the values were within the historical control limits.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No further details available.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There was an inconsistent pattern of food consumption. The findings were not adverse.
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
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester and 1540-5620 mg/kg bw/day phytosterol
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Pup mortality was increased in the mid and high dose groups compared with the controls; however, the values fell within the historical control limits.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 8.1 other: % (w/w) phytosterol ester; equivalent to 2500-9100 mg/kg bw/day phytosterol ester and 1540-5620 mg/kg bw/day phytosterol
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Table 1 Intake of phytosterol ester in parent animals

                      Phytosterol esters (% in diet)
 Generation  Sex  Period  1.6  3.2  8.1
             Phytosterol ester (g/kg bw/day)
 F0 Males  Premating weeks 1 -10  0.9 -0.5  1.8 -1.6  4.4 -2.7
  Females  Premating weeks 1 -10  1.1 -0.6  2.1 -1.3  5.6 -3.4
     Gestation week 1  0.8 1.5  3.8 
     Gestation week 2  0.8  1.5  3.9
     Gestation week 3  0.5  1.0 2.5 
     Lactation week 1  1.1  2.1  5.7
     Lactation week 2  1.8  3.5 9.1 
   Lactation week 3*  2.3  4.5  12.6
 F1  Males  Premating weeks 1 -10  1.3 -0.6  2.6 -1.1  6.2 -2.8
   Females  Premating weeks 1 -10  1.3 -0.6  2.6 -1.2  6.5 -3.3
     Gestation week 1  0.7  1.4 3.6 
     Gestation week 2  0.7  1.4  3.6
     Gestation week 3  0.5  0.9  2.3
     Lactation week 1  1.0  2.1  4.8
     Lactation week 2  1.7  3.4  8.5
     Lactation week 3*  2.1  4.4  10.9

* As pups started to eat on approximately post natal day 14, test substance intake in lactation week 3 is unrealistic.

Values are group means; during premating, n=7 cages/group/sex; during gestation and lactation, n=19 -27 female rats/group.

Table 2 Reproductive performance of female rats

              Phytosterol esters (% of diet)
 Parameter  Generation  0  1.6  3.2  8.1
 Mating index (%)1  F0  100 100  100  100 
   F1 100  100  100  96
  Fertility index (%)2  F0  100 96 86 93 
  F1   96  93  96  89
 Fecundity index (%)3  F0  100 96  86  93 
   F1 96   93  96  93
 Gestation index (%)4  F0  100  100  96  96
   F1  100  100  96  100
 Precoital time (days)5  F0  3.3 ± 0.65  3.7 ± 0.73  2.6 ± 0.23  2.4 ± 0.54
   F1  2.4 ± 0.28  3.3 ± 0.51  2.6 ± 0.20  3.6 ± 0.63
 Gestation time (days)5  F0  21.2 ± 0.11  21.4 ± 0.12 21.3 ±  0.14  21.4 ± 0.12
   F1  21.2 ± 0.12  21.4 ± 0.11  21.4 ± 0.11  21.2 ± 0.11
 Post implantation loss/animal (%)5,6  F0 15.31 ± 4.24   19.01 ± 3.83  19.33 ± 5.29  21.66 ± 5.18
   F1  6.44 ± 1.43  13.05 ± 2.21  11.21 ± 3.78  10.81 ± 1.54

1 (no. of females mated/no. of females placed with males) X 100

2 (no. of females pregnant/no. of females placed with males) X 100

3 (no. of females pregnant/no. of females mated) X 100

4 (no. of females with live pups/no. of females pregnant) X 100

5 Values are means ± SEM; statistical test: Kruskal-Wallis + Mann-Whitney U test

6 Total post implantation loss = ((no. of implantation sites/no. of pups born alive)/no. of implantation sites) X 100

Table 3 Summary of offspring data

              Phytosterol esters (% of diet)
 Parameter  Generation  0  1.6  3.2  8.1
 Pups delivered (total)1  F0 10.39 ± 0.37   10.26 ± 0.44 10.33 ± 0.48  9.68 ± 0.49
   F1  10.56 ± 0.28  9.73 ± 0.44  9.96 ± 0.40  10.60 ± 0.27
 Live birth index (%)2,3 F0   92  92  92 93 
   F1  100  98  99  98
 Pup mortality day 1 (%)2  F0 8.2   8.3 8.5  7.0 
   F1  0  1.6  1.5  1.5
 Viability index day 4 (%)2,4  F0  93.3  87.4*  84.6**  84.0**
   F1  98.9  97.2  95.8*  89.7***
 Viability index day 21 (%)2,5  F0  98  100  100  99
   F1  99  99  99  100
 Dams with no deaths, day 216  F0  20  17  13  14
   F1  21  21  20  18
 Dams with 1 or more deaths, day 216  F0  7  10  11  11
   F1  6  5  7  7
 Whole litter losses2  F0  3  3  2  5
   F1  0  0  1  0
 Sex ratio day 1 (%)7  F0  51  53  52  49
   F1  53  50  48  60

1 Values are means ± SEM per litter, statistical test: Kruskal-Wallis + Mann-Whitney U test

2 Statistical test: Fisher's exact test (*P<0.05; **P<0.01; ***P<0.001)

3 (no. of pups born alive/no. of pups born) X 100

4 (no. of pups at day 4/no. of live pups at day 1) X 100

5 (no. of pups at day 21/no. of live pups at day 4) X 100

6 Litter data analysed by Kruskal-Wallis

7 % of male pups of total live pups at day 1

Applicant's summary and conclusion

Conclusions:
In a dietary two-generation reproductive toxicity study conducted using a protocol comparable with OECD 416 and in compliance with GLP (reliability 1) no adverse effects were reported for general systemic toxicity, or reproductive and developmental toxicity in any generation. The highest concentration tested was 8.1% (w/w) phytosterol ester in diet. This gave a NOAEL of 2500-9100 mg/kg bw/day phytosterol ester or 1540-5620 mg/kg bw/day phytosterol.