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Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

The available weight of evidence from studies conducted on substances representative of the different constituents (glycerides, fatty acids, tocopherols, sterols, squalene and hydrocarbons) indicated that the test substance is not likely to be a reproductive toxicant.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 90 d oral repeated dose study was conducted in F344/N rat to evaluate the sub-chronic toxicity of castor oil in which reproductive parameters were also screened. Rats (10/sex/group) were treated for 13 weeks with 0, 0.62, 1.25, 2.5, 5.0 or 10% castor oil mixed in diet. Apart from the standard sub-chronic toxicity examinations, sperm count, motility and morphology were evaluated at necropsy and vaginal cytology during the week preceding necropsy. Male and female gonadal weights were also determined with gross pathology and histopathology of reproductive organs at termination.
GLP compliance:
yes
Remarks:
FDA Good Laboratory Practices Regulations (21 CFR 58)
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA
- Age at study initiation: 6 wk
- Fasting period before study: No
- Housing: 5 per cage in polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets. The cages were stored on stainless steel racks equipped with an automatic watering system
- Diet: NIH 07; available ad libitum
- Water: Ad libitum
- Acclimation period: 14 d
- Other: Feed hoppers in the animal cages were changed twice weekly


ENVIRONMENTAL CONDITIONS
- Temperature: 68-76°F
- Humidity: 42-72%
- Air changes: 10/h
- Photoperiod: 12 h dark/12 h light
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Method of mixing: Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amounts of feed in a twin-shell blender and blended for 15 min to achieve a uniform mix.
- Storage temperature of food: Stored for no longer than 3 weeks at 5°C
- Stability under test conditions: 0.5% dose level is stable for at least 21 d when stored in the dark at 5°C and for 3 d when stored open to air and light in a rodent cage.
Details on mating procedure:
No mating performed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determined by HPLC at the study and analytical chemistry laboratories in duplicates. The results of the analyses for all dose mixtures given to the animals ranged from 97% to 106% of the target concentrations.
Duration of treatment / exposure:
13 wk or 90 d

Frequency of treatment:
Daily

Details on study schedule:
None
Remarks:
Doses / Concentrations:
0, 0.62, 1.25, 2.5, 5.0 or 10% in diet
Basis:
actual ingested
No. of animals per sex per dose:
10 rats per sex per dose
Control animals:
yes, plain diet
Details on study design:
None
Positive control:
Not applicable
Parental animals: Observations and examinations:
All routine examinations were performed including body weight, food consumption, hematology and clinical chemistry. For details see section 7.5.1: Repeated dose toxicity-oral; Endpoint study record: Castor oil (232-293-8), Irwin, 1992.
Oestrous cyclicity (parental animals):
Yes (at 12 wk)
Sperm parameters (parental animals):
Testis weight, epididymis weight, sperm count, motility and morphology were evaluated at necropsy (termination of study)
Litter observations:
Not examined
Postmortem examinations (parental animals):
Following reproductive organs were examined grossly and histologically apart from routine examinations:
Epididymis/seminal vesicles/prostate/testes or ovaries/uterus

- Complete histopathology examinations were conducted on all rats from the control and 10% dose groups
Postmortem examinations (offspring):
Not examined
Statistics:
Statistically analyzed within each sex by one-way Analysis of Variance tests, followed by Dunnett's t-test for pair-wise comparisons (p < 0.05)
Reproductive indices:
No data
Offspring viability indices:
Not calculated
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
No significant changes were noted for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of female estrous cycles. No significant changes were noted in male and female gonadal organs at necropsy except there was a slight decrease in epididymal weight (6-7%) which occurred in the middle- and high-dose groups, but this was not dose-related.
Key result
Dose descriptor:
NOAEL
Effect level:
> 10 other: % in diet (i.e. ca. 5,800 mg/kg bw/d) (male/female)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant changes for male reproductive endpoints, including sperm count and motility, and no changes in length of female estrous cycles
Key result
Critical effects observed:
no
Not applicable
Key result
Dose descriptor:
NOAEL
Remarks on result:
not measured/tested
Reproductive effects observed:
no

None

Conclusions:
Under the conditions of this study, the NOAEL of castor oil for reproductive toxicity screening in rats was determined to be 10% in diet (i.e. 5800 mg/kg bw/day based on actual feed consumption and body weight data).
Executive summary:

A 90 d oral repeated dose study was conducted in F344/N rats to evaluate the sub-chronic toxicity of the constituent castor oil in which reproductive parameters were also screened. Rats (10/sex/group) were exposed for 13 weeks to 0, 0.62, 1.25, 2.5, 5.0 or 10% castor oil mixed in diet. Apart from the standard sub-chronic toxicity examinations, sperm count, motility and morphology were evaluated at necropsy and vaginal cytology during the week preceding necropsy. Male and female gonadal weights were also determined with gross pathology and histopathology of reproductive organs at termination. No significant changes were noted in a screening for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of female estrous cycles. No significant changes were noted in male and female gonadal organs at necropsy except there was a slight decrease in epididymal weight (6-7%) which occurred in the middle- and high-dose groups, but this was not dose-related. Under the conditions of this study, the NOAEL of castor oil for reproductive toxicity screening in rats was determined to be 10% in diet (i.e. 5800 mg/kg bw/day based on actual feed consumption and body weight data) (Irwin, 1992).

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1977
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A 13 wks (90 d) oral combined repeated dose and reproduction/developmental screening study was conducted in Sprague Dawley rat to evaluate the reproductive parameters. After 10 weeks of treatment with 15% crude palm oil, 10 males and 20 females (15 - 16 weeks of age) were mated for 18 d. The females were then allowed to produce young. Maternal bodyweight and reproductive parameters (e.g. % implantation, % surviving young, % embryo loss, ...) were recorded. At 5 weeks of age, the young were sacrificed. One male and 2 females from each litter was sacrificed. Liver and kidneys were weighed. For 5 males and 5 females selected randomly, these organs were examined microscopically.


GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Own breeding
- Age at study initiation: ca. 6 weeks
- Weight at study initiation: 150 g
- Housing: 5 per sex per cage
- Diet (e.g. ad libitum): 40% wheat, 20% maize, 12% fish meal, 7% blood powder, 15% oil and 6% vitamin/minerals complement
- Water (e.g. ad libitum): yes
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): every 4 - 5 d
- Storage temperature of food: 4°C
Details on mating procedure:
After 10 weeks of treatment, 10 males and 20 females (15 - 16 weeks of age) were mated for 18 d. The females were then allowed to produce young.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
13 wks or 90 d
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
15%
Basis:
nominal in diet
No. of animals per sex per dose:
10 males, 20 females for the reproduction screening
Details on study design:
No data
Positive control:
No data
Parental animals: Observations and examinations:
REPRODUCTION SCREENING:
Maternal bodyweight and reproductive parameters (e.g. % implantation, % surviving young, % embryo loss etc) were recorded. At 5 weeks of age, the young were sacrificed. One male and 2 females from each litter was sacrificed. Liver and kidneys were weighed. For 5 males and 5 females selected randomly, these organs were examined microscopically.
Postmortem examinations (offspring):
Microscopic pathology of liver and kidney of young rats were performed from the reproduction screening at 35 d of age.


Statistics:
No data
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Reproductive performance:
no effects observed
During the reproductive screening study, no effects on maternal bodyweight evolution, reproductive parameters, pup liver and kidney weights, and pup liver and kidney histopathology

Key result
Dose descriptor:
NOAEL
Effect level:
> 15 other: % in diet (i.e., equivalent to 17,000 - 7,000 mg/kg bw/day as the bodyweight of animals increased regularly over the course of the study)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No significant toxicity on maternal rats or pups
Key result
Critical effects observed:
no
During the reproductive screening study, no effects were observed on maternal body weight, reproductive parameters, pup liver and kidney weights, and pup liver and kidney histopathology.

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
15 other: % w/w in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
Under the conditions of this study, the NOAEL of the substance can be considered to be 15% in diet, as no significant toxicity were observed on maternal animals and pups.
Executive summary:

A combined repeated and reproductive screening study was conducted to determine the effect of the constituent ‘glycerides, C16-18 and C18-unsatd.’ (as crude palm oil) on rats when administered for 13 weeks at 15% in diet. Results were compared to those obtained with heated palm oil, crude/heated soy oil, crude/heated peanut oil, or crude/heated sunflower oil at the same concentration. Clinical signs and bodyweight were recorded throughout the study. After 13 weeks, hematology, clinical chemistry and urinalysis parameters were assessed, as well as gross and microscopic pathology. After 10 weeks of treatment, 10 males and 20 females were mated for 18 d. Maternal bodyweight and reproductive parameters were recorded. At 5 weeks of age, the young were sacrificed. Liver and kidneys weights were recorded and these organs were examined microscopically. The substance did not show any adverse effects on male and female rats compared to other crude or heated vegetable oils when administered for 13 weeks at 15% in diet. Furthermore, no signs of toxicity were observed on maternal rats or pups in the follow-up reproductive screening trial. Under the conditions of this study, the NOAEL of the substance can be considered to be 15% in diet as no significant toxicity were observed on maternal rats and pups (Coquet, 1977).

Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Multigeneration breeding studies in rats were conducted with the constituent red palm oil according to the protocol used in earlier studies on unconventional oils (Rukmini, 1988 and Rukmini, 1990) according to the guidelines of the FDA/WHO/DGHS safety evaluation protocol (FDA, 1970).

Groups of 24 (12 male and 12 female) inbred weanling albino rats were given a 20% protein diet, adequate in all nutrients and containing 10% of either groundnut oil (GNO; controls), red palm oil (RPO), refined, bleached, deodorized palm olein oil (RBDPO), or hydrogenated vegetable oil (HVPO) with 30% mahua oil.

Bodyweight and food intake were recorded weekly for 15 weeks (100 - 120 days).

Propagation of the three generations was conducted as follows: F0 rats were mated twice to produce F1a and F1B litters. F1b rats were mated twice to produce F2a and F2b litters, and two matings of the F2b rats produced the F3a and F3b litters. The parameters recorded included fertility index or conception rate, sex ratio, mean weaning weight, pre-weaning mortality, number of days from introduction of mating, behaviour of pups and adults.
GLP compliance:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
The rats were kept in galvanized aluminium cages at a constant temperature of 25°C, with 50-60% relative humidity and a 12 h light/dark cycle. The rats were given a 20% protein diet adequate in all nutrients. Food and water were given ad libitum.
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Group 1 (control): 10% groundnut oil
Group 2: 10% red palm oil
Group 3: 10% bleached and deodorized palm olein
Group 4: 10% hydrogenated oil containing 30% of mahua oil

The substances were mixed in the diet.
Details on mating procedure:
12 males and 12 females in each group were mated after 100-120 d. The mated animals were allowed to be together for a period of 18-20 d. Mating behaviour was checked in the morning by looking for the capulatory plug in the bottom tray to confirm for pregnancy. After further confirmation by palpation and periodic checking for the increase in body weight in females, the mated animals were separated and housed in individual plastic cages.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
None
Duration of treatment / exposure:
3 generations
Frequency of treatment:
Daily
Details on study schedule:
The F0 rats were mated twice to produce the F1a and F1b litters. The F1b rats were mated twice to produce the F2a and F2b litters, and two matings of the F2b rats produced the F3a and F3b litters.

Once the female delivered, the litters were counted, sexed and weighted and return to the mother where they staid for the lactation period of 21 days. After the 21 days they were taken from the mothers, the number of female and males were recorded and their weight. Six male and six female pups of the F1a generation were killed after weaning. Gross pathology and organ weights were recorded. The mothers got to rest for 1 week and then again housed with the same males. This second mating produced F1b pups. After weaning the F1b pups were fed for 90 days, allowed to mature and bred to produce a second generation (F2b rats).

Remarks:
Doses / Concentrations:
10%
Basis:
nominal in diet
No. of animals per sex per dose:
12
Control animals:
yes
Details on study design:
The toxic or non-toxic responses from the reproduction studies were expressed in terms of indices that considered all stages from conception to weaning (as developed by Mirone L, Panzarella FP and Cerecodo LR, 1948. A new method of reporting data on reproduction and lactation in the mouse. Science,108:139).
Positive control:
None
Parental animals: Observations and examinations:
Bodyweight and food consumption, recorded weekly
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
The sex ratio, mean weaning weight, pre-weaning mortality and behaviour such as righting reflex (tested on Day 2 after birth), negative geotaxis (tested on Day 5 after birth), cliff drop aversion (tested on Day 5 after birth) and auditory startle response (tested on Day 12 after birth).
Postmortem examinations (parental animals):
The organs of all adults of each generation were weighed and the relative body weight were calculated.
Postmortem examinations (offspring):
Gross pathology and organ weight.
Statistics:
Analyses of variance was used to test the difference between groups for parametric data and the Chi-square test was used for non-parametric data.
Reproductive indices:
Fertility Index (FI)
Number of days from introduction for mating (IFM)
Behaviour of adults
Offspring viability indices:
Sex ratio
Mean weaning weight
Preweaning mortality
Behaviour of pups
Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
No significant differences were found in body weight gain between groups in any of the three generations. The food consumption and weight gain of rats fed crude palm oil was similar to that of the controls and they also showed an adequate growth and development. In the first generation 100% of matings resulted in pregnancies in both the first and the second mating. In the second generation 91.6% of dams conceived in both matings, and in the third generation 100% and 91.6% conceived in the first and second matings, respectively.

No abnormal behavioural or reflexological changes in any of the animals.

No significant changes attributable red palm oil were evident in the relative weights of the liver, heart, spleen, lungs, kidney, testes/ovaries or total body weight in the F0 generation rats.

Gross pathology indicates no adverse abnormalities in the organs.
Key result
Dose descriptor:
NOAEL
Effect level:
10 other: % in diet
Sex:
male/female
Basis for effect level:
other: no significant adverse effect were observed on any of the reproductive or toxicological parameters
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Effect level:
10 other: % w/w in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effects
Key result
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Mean litter size, birth and weaning weights were comparable and were not found to be significantly different in all the three generations of all groups.

The sex ratio at birth and weaning did not indicate any difference in the relative fitness of each sex with maturity. Pre-weaning mortality was higher in the first generation animals of both matings in all four groups, except for the second mating of the groups, which showed comparatively lower percentages. In the second mating of the second generation, the group showed a comparatively high percentage, and in the second mating of the third generation, percentages were higher for all groups. These mortalities are not likely to be treatment-related because the controls also had high pre-weaning mortality rates. Cannibalism was observed in some animals and the mortality of pups may be mainly due to this factor or to maternal negligence, rather than to the presence of any toxic substances that may have been excreted in milk or consumed by the pups.

There was no abnormality or unusual delay in conception or delivery in the groups.

No abnormal behavioural or reflexological changes in any of the animals.

No significant changes attributable to red palm oil were evident in the relative weights of the liver, heart, spleen, lungs, kidney, testes/ovaries or total body weight in the F1b or F2b generation rats.

Gross pathology indicates no adverse abnormalities in the organs.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 other: % w/w in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effects
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
10 other: % w/w in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically significant effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
In this multigeneration study no adverse effects were observed on the reproductive or toxicological parameters studied. According to the author, these results indicate that the substance can be safely used as an edible oil.
Executive summary:

A three-generation study was conducted with groups of 12 male and 12 female rats fed 10% of either groundnut oil (controls), the constituent ‘glycerides, C16 -18 and C18 -unsatd.’ (as red palm oil), refined, bleached and deodorized palm olein or hydrogenated vegetable oil containing mahua oil through diet. Reproduction parameters including percentage conception, birth, weight, litter size, weanling weight, sex ratio at birth and weaning, preweaning mortality and number of days from introduction to mating, were recorded. Behavioural and reflexological tests were conducted on preweaning animals. No significant differences were recorded in any of the reproductive or toxicological parameters observed between the treated and control groups. According to the authors, these results indicate that the substance can be safely used as an edible oil (Manorama, 1993).

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
A two generation reproductive toxicity study was conducted in Mongolian gerbils to assess the effects of dietary exposure to 8.75% w/w of the constituent palm kernel oil (PO). 5 couples of adult gebils with their sucklings (generation 1) were assigned to various diteary groups: palm kernel oil (8.75% w/w), sunflower seed oil (8.75% w/w) or cholesterol (0.2% w/w). The ensuing generations were put on the same diets as their parents with the exception of the cholesterol-containing diets which was reduced to 0.05%. After 4 months, 4 adults in each group were terminated and histopathological changes in liver and spleen were observed along with liver and serum cholesterol levels. In the second generation parameters such as frequency of litters, number of pups, mean litter size, mean weight and postnatal mortality were also observed.
GLP compliance:
not specified
Species:
other: gerbil
Strain:
other: Mongolian
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Adult gerbils and their sucklings (F1) were used at the initiation
- Housing: Housed in couples in standard laboratory cages with a wire mesh lid with food hopper. The bedding consisted of wood shavings.
- Diet (e.g. ad libitum): Diets manufactured by Hope Farms BV (Woerden, The Netherlands) were used. The test diet contained increased protein (26%), biotin, choline, inositol and vitamin E (50%) and cellulose (1%).
- Water (e.g. ad libitum): Water acidified with HCI (pH 3), ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 20°C
- Humidity: 50%
- Photoperiod: 10 h light/14 h dark


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Not reported



Details on mating procedure:
No data
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Duration of treatment / exposure:
2 generations (specific period not mentioned)
Frequency of treatment:
Daily
Details on study schedule:
Not reported
Remarks:
Doses / Concentrations:
8.75% w/w in basic diet
Basis:
nominal conc.
No. of animals per sex per dose:
5 couples per group
Control animals:
yes, plain diet
Details on study design:
No data
Positive control:
Not applicable
Parental animals: Observations and examinations:
Not examined
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F2] offspring: Frequency of litters, number of pups, mean litter size, mean weight at 6 months of age and postnatal mortality




Postmortem examinations (parental animals):
Not examined
Postmortem examinations (offspring):
SACRIFICE
- Four adult animals out of each dietary group of the F1 generation were sacrificed after 4 months.


HISTOPATHOLOGY / ORGAN WEIGHTS
- Tissues/organs examined: Liver and spleen
- Brief description on slide preparation: After fixation and paraffin embedding of parts of the liver and spleen, 4-µm thick sections were cut for analysis, followed by staining with hemaloxylin, eosin and Gomori's reticulin.


CLINICAL CHEMISTRY
- Total serum cholesterol and liver cholesterol content were also determined along with histopathological parameters in F1 generation after sacrifice
Statistics:
The Student-t test for unpaired samples was used to compare the variables of the different groups. For large differences in variance between the samples, the Wilcoxon test for unpaired samples was used. p < 0.05 was considered significant.
Reproductive indices:
Not reported
Offspring viability indices:
Not reported
No data


Key result
Dose descriptor:
NOAEL
Effect level:
8.75 other: % w/w in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant treatment-related effect
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Effect level:
8.75 other: % w/w in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant treatment-related effect
Key result
Critical effects observed:
no
Clinical signs:
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
no effects observed
BODY WEIGHT (OFFSPRING): No significant difference compared to controls was observed (F1 and F2)


HISTOPATHOLOGY (F1): The livers and spleens of the animals on diets basal diet and palm kernel oil diet had a normal histology.


CLINICAL CHEMISTRY (F1):
- Serum cholesterol (mmol/L): Slight increase in PO fed group (3.9±0.5) in comparison with control (2.8±0.3).
- Liver cholesterol content: No significant difference was observed.


FREQUENCY OF LITTERS (F2): No significant effects were observed.


NUMBER OF PUPS (F2): No significant difference was observed (PO: 116 and basal diet (control): 146).


MEAN LITTER SIZE (F2): No significant difference was observed (PO: 4.7±1.7 and basal diet (control): 4.9±1.6).


POSTNATAL MORTALITY (F2): No significant mortality was observed in comparision to basal diet group ( PO: 4 % and basal diet (control): 3 %)





Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
8.75 other: % w/w in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no significant treatment-related effect
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
> 8.75 other: % w/w (i.e. ca. 4,375 mg/kg bw/day)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on frequency of litters, mean litter size, total no. of newborns and no suckling death
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
The test substance, exhibited no significant reproductive toxicity at 8.57% concentration level in a two generation study.
Executive summary:

A two generation reproductive toxicity study was conducted in Mongolian gerbils to assess the effect of the constituent ‘glycerides, C8-18 and C18-unsatd.’ (as palm kernel oil). Adult Mongolian gerbils (highly resistant to atherosclerosis) and their sucklings (first generation) were randomly assigned to two groups of 5 couples each (group 1: basal diet; group 2: 8.75% test substance in diet). Four months after starting the diets, four adult animals of each dietary group of the first generation were sacrificed and histopathological changes in liver and spleen were observed along with liver and serum cholesterol levels. In the second generation parameters such as frequency of litters, number of pups, mean litter size, mean weight, postnatal mortality were also observed. Both in the first and second generations, no significant differences of body weight were observed compared to controls. The liver and spleen of the animals of the first generation also had a normal histology. However, a slightly elevated serum cholesterol level was observed in the test substance fed group. No significant effect on frequency of litters, mean litter size, total no. of newborns, and no suckling death were found in the second generation of the test substance fed group compared to the basal dietary group. Hence, under the test conditions, the substance did not exhibit any significant reproductive toxicity at 8.57% in diet (Temmerman, 1988).

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A reproductive toxicity study was conducted in Wistar rats to assess the effect of the constituent alpha-tocopherol. Fertility parameters such as pregnancy rates, vaiability of foetus, number of resorptions and developmental parameters such as effects on ossification and fetal weights were observed.
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on mating procedure:
Not reported
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
20 d prior to mating and during gestation
Frequency of treatment:
Daily
Details on study schedule:
Not reported
Remarks:
Doses / Concentrations:
0.75 (control), 7.5, or 75 mg/day alpha-tocopherol (equivalent to 2.14, 21.4 and 214.2 mg/kg bw/day, assuming the average rat body weight as 0.35 kg)
Basis:
nominal in diet
No. of animals per sex per dose:
10 male and female Wistar rats/dose
Control animals:
yes
Key result
Dose descriptor:
LOAEL
Effect level:
21 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Decreased pregnancy rates (50% and 90% at 21 and 214 mg/kg bw/day respectively), few resorptions and mild cases of ossification were observed at both tested doses.
Key result
Critical effects observed:
not specified
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
214 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
Key result
Reproductive effects observed:
not specified

Details of results:

- The pregnancy rate for the animals of the 21 and 214 mg/kg bw/day groups were 90% and 50%, respectively.

- The number of viable fetuses was slightly less in the high dose group compared to controls.

- A few resorptions were observed for both test groups.

- Fetal weight was similar for all groups.

- Very mild cases of ossification retardation were observed. No abnormal fetuses were reported.

Conclusions:
Under the test conditions, the LOAEL of alpha- tocopherol for reproductive toxicity in rats was determined to be 21 mg/kg bw/day.
Executive summary:

A reproductive toxicity study was conducted in Wistar rats to assess the effect of the constituent alpha-tocopherol in rats. Groups of 10 male and female Wistar rats were fed a diet containing 0.75 (control), 7.5, or 75 mg/day of the substance (i.e. equivalent to 2.14, 21.4 and 214.2 mg/kg bw/day, assuming the average rat body weight as 0.35 kg) 20 d prior to mating and during gestation. Gravid animals were killed on Day 20 of gestation. All of the animals given control feed were gravid, but the pregnancy rate for the animals of the 21and 214 mg/kg bw/day groups were 90% and 50%, respectively. The number of viable fetuses was slightly less in the 214 mg/day group compared to controls. A few resorptions were observed for both test groups. Fetal weight was similar for all groups. Very mild cases of ossification retardation were observed. No abnormal fetuses were reported. The researchers concluded the test substane to have "little effect on the rat fetus" (Fiume, 2002). However,the toxicological relevance of the observedeffects could not be evaluated due to limited details. Further, are cent Fiume (CIR) report from 2014 indicated absence of reproductive and development effects up to the highest tested dose i.e., 241 mg/kg bw/day.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
The effects on reproductive indices were investigated on dietary exposure to 0, 0.002, 0.2, & 2% (i.e., approx. 1.02, 103 and 1028 mg/kg bw/day) d-alpha-tocopherol (polyethylene glycol) 1000 succinate (TPGS).
GLP compliance:
no
Limit test:
no
Justification for study design:
- The animals were mated on Day 112 of treatment to produce the F1a generation and on Day 175 to produce the F1b generation. The F0 animals were maintained on their respective diets for 265 d of treatment, then sacrificed and examined histopathologically.
Species:
rat
Strain:
other: COBS, CD, albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: five per cage in suspended wire-bottom cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
No data
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged: the male was removed from the cage, a nesting pan was introduced and the female was allowed to litter
- Any other deviations from standard protocol: no data
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
The parent generation was treated for 264-268 days. All offspring were killed after 5 weeks of weaning.
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations: 0, 0.002, 0.2, & 2% [i.e., (1) approx. 1.02, 103 and 1028 mg/kg bw/day, assuming 0.35 kg as average weight of adult female rats and 18 g as daily food consumption (2) approx. 0.8, 80 and 800 mg/kg bw/day, assuming 0.50 kg as average weight of adult female rats and 20 g as daily food consumption]
No. of animals per sex per dose:
15/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment: random
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: no data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: no data

BODY WEIGHT: Yes
- Time schedule for examinations: prior to treatment, twice during the first week of feeding and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/rat/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
No data
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
insemination, fertility, gestation, viability, lactation, average gestation (days), average litter size, sex (M/F), mean mortality at birth, mean mortality 0-8 weeks


GROSS EXAMINATION OF DEAD PUPS:
No data
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 265-268 days of treatment when it was certain no additional matings were necessary.
- Maternal animals: All surviving animals after 265-268 days of treatment when it was certain no additional matings were necessary.

GROSS NECROPSY
- Gross necropsy consisted of: no data

HISTOPATHOLOGY / ORGAN WEIGHTS
organ weights, hematocrit, hemoglobin concentration, total and differential white cell counts, serum glutamic oxalacetic transaminase, serum alkaline phosphatase, urea nitrogen, lactic acid dehydrogenase, serum glucose, serum total protein, triglyceride and cholesterol.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5 weeks after weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of: no data

HISTOPATHOLOGY / ORGAN WEIGTHS
No data
Statistics:
Data in all studies were analyzed statistically by analysis of variance, Duncan's multiple range ste, or Student's T-test p<0.05.
Reproductive indices:
Reproductive indices such as mean gestation period, litter size, sex ratio, and mortality of pups or parents were observed
Offspring viability indices:
Yes
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
Reproductive indices (mean gestation period, litter size, sex ratio, and mortality of pups or parents) were unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
> 1 028 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects on reproductive indices, clinical chemical and haematological parameters
Key result
Dose descriptor:
NOAEL
Effect level:
> 800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No effects on reproductive indices, clinical chemical and haematological parameters
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 028 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no treatment-related effects
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No treatment related effects
Reproductive effects observed:
not specified

No apparent differences in reproductive indices were seen between controls and treated groups of parents. Mean gestation period, litter size, sex ratio, and mortality of pups and parents were unaffected by the test substance. Hematology and clinical chemistry done after 255 days of treatment revealed no toxicologic differences between control and high dose group. None of the organ weights (neither absolute nor relative) of the treated groups were significantly different from control. Microscopic examination of tissues of high dose and control F0 and F1b animals revealed no morphological changes that were attributable to ingestion of the test substance. Ingestion of the test substance had no effect on body weight gain of the pups.

Conclusions:
Under the study conditions, the NOAEL of the substance was determined to be 2% (800 mg/kg bw/day for male and 1028 mg/kg bw/day for female).
Executive summary:

The dietary effects on reproductive indices were investigated on exposure to d-alpha-tocopherol (polyethylene glycol) 1000 succinate (TPGS). Groups of 15 COBS CD Albino rats of each sex were fed diets containing the substance at concentrations of 0, 0.002, 0.2, or 2% (i.e. approx. 0.8 -1.02, 80-102.8, 800-1028.4 mg/kg bw/day, considering average adult rat bw 0.500 kg for male and 0.350 kg for female, food intake 20 g/day for male and 18 g/day for female) for 264 to 268 d. The animals were mated on Day 112 of treatment to produce the F1a generation and on Day 175 to produce the F1b generation. The F0 animals were maintained on their respective diets to 266 to 268 d of treatment, then sacrificed and examined histopathologically. Reproductive indices (mean gestation period, litter size, sex ratio, and mortality of pups or parents) were unaffected by treatment. Clinical chemical and haematological parameters were normal in the F0 generation 10 d before terminal sacrifice. None of the organ weights (neither absolute nor relative) of the treated groups were significantly different from control. Microscopic examination of tissues of high dose and control F0 and F1b animals revealed no morphological changes that were attributable to ingestion of the test substance. Ingestion of the test substance had no effect on body weight gain of the pups. Under the study conditions, the NOAEL of the substance was determined to be 2% (800 mg/kg bw/day for male and 1028 mg/kg bw/day for female) (JECFA, 1987).

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Secondary literature source with limited details but used in the Integrated Laboratory Systems (ILS) assessment report
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Adult male albino Wistar rats exposed to β-sitosterol at 0.5 or 5 mg/kg bw/day (0.001 or 0.012 mmol/kg bw/day) for 16, 32 and 48 d respectively with a withdrawl period of 30 d were evaluated for fertility and reproductive parameters.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
No data
Route of administration:
subcutaneous
Vehicle:
not specified
Details on exposure:
No data
Details on mating procedure:
Not applicable
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
Low and high dose groups: Administered for 16, 32 and 48 d respectively.
Withdrawal group: Administered for 16, 32 and 48 d and then treatment was withdrawn for 30 d.
Frequency of treatment:
Daily
Details on study schedule:
Not applicable
Remarks:
Doses / Concentrations:
0.5 or 5 mg/kg bw/day (0.001 or 0.012 mmol/kg/day),
Basis:
nominal conc.
No. of animals per sex per dose:
10/dose
Details on study design:
No data
Positive control:
No data
Parental animals: Observations and examinations:
No data
Oestrous cyclicity (parental animals):
Not applicable
Sperm parameters (parental animals):
Not applicable
Litter observations:
Not applicable
Postmortem examinations (parental animals):
No data
Postmortem examinations (offspring):
Not applicable
Statistics:
No data
Reproductive indices:
Fertility, sperm concentrations and testicular weight were determined.
Offspring viability indices:
Not applicable
Clinical signs:
not specified
Body weight and weight changes:
not specified
Food consumption and compound intake (if feeding study):
not specified
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
not specified
Other effects:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related
REPRODUCTIVE FUNCTION: SPERM MEASURES:
At 0.5 mg/kg bw/d β –sitosterol (0.00121 mmol/kg bw/d) there was significant decreased sperm concentrations after 48 d of treatment.
At 5 mg/kg bw/day (0.0121 mmol/kg bw/day) β –sitosterol, sperm concentrations were reduced after 16, 32, and 48 d of exposure.


REPRODUCTIVE PERFORMANCE
Fertility: At 5 mg/kg/day (0.0121 mmol/kg/day) β –sitosterol, fertility was reduced after 42 and 48 d of exposure.

Testicular weight:
At 0.5 mg/kg/day β –sitosterol ( 0.00121 mmol/kg/day) testicular weight was decreased after 32 and 48 d of treatment.
At 5 mg/kg testicular weight decreased in a time-dependent manner.

Withdrawal from treatment for 30 d did not restore sperm count or testicular weight.
Key result
Dose descriptor:
LOAEL
Effect level:
0.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: At 0.5 mg/kg bw/day there was a significant decrease in sperm concentration and testicular weight. At 5 mg/kg bw/day decreased fertility was observed in addition to other effects seen at low dose.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
0.5 mg/kg bw/day (nominal)
System:
male reproductive system
Organ:
testes
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Dose descriptor:
NOAEL
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified

None

Conclusions:
Under the conditions of this study, the LOAEL of the substance in male rats was determined to be 0.5  mg/kg bw/day, based on decreased sperm concentration and testicular weight at lowest tested dose.
Executive summary:

A reproductive toxicity study was conducted through the sub-cutaneous route in male albino Wistar rats to assess the effect of the constituent β-sitosterol on reproductive performance in males. Adult male albino Wistar rats exposed to the substance at 0.5 or 5 mg/kg bw/day (0.001 or 0.012 mmol/kg bw/day) for 16, 32 and 48 d respectively (with a withdrawl period of 30 d) were evaluated for fertility and reproductive parameters. At 0.5 mg/kg bw/day, there was significantly decreased sperm concentrations and decreased testicular weight. At 5 mg/kg bw/day, fertility was reduced in addition to effects observed at lower dose. Under the conditions of this study, the LOAEL of the substance in male rats was determined to be 0.5  mg/kg bw/day, based on decreased sperm concentration and testicular weight at lowest tested dose (Tice, 1997).

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Secondary literature source with limited details, but used in the Australia New Zealand Food Authority (ANZFA) assessment of phytosterol esters, and followed GLP
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A two generation reproductive toxicity study was conducted in Wistar rats to assess the dietary effects of 0, 1, 2 and 5% of the constituent plant sterol; equivalent to 0, 500–1300 mg/kg bw/day (low dose), 1000-2600 mg/kg bw/day (mid-dose) and 2800-4400 mg/kg bw/day (high dose)). Treatments began in F0 animals 10 weeks before mating and continued until weaning (3 weeks post parturition) when F0 males were sacrificed. F0 females were maintained for another 3 weeks and then sacrificed. During this entire period, various parameters were examined. Selected pups of F1 generation were treated with plant sterols in diet at the same levels as their F0 parents for 10 weeks pre-mating. The treatment, observation and sacrifice schedule followed that of the F0 generation. F1 and F2 litters were examined at 1, 4, 7, 14 and 21 d (weaning).
GLP compliance:
yes
Remarks:
Lab name not reported in the ANZFA risk analysis report
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld
Route of administration:
oral: feed
Vehicle:
not specified
Details on exposure:
Test substance intake was monitored:
At pre-mating and gestation sterol doses in males and females of F0 and F1 generation were in the ranges:
500-1300 mg/kg bw/day (low dose)
1000-2600 mg/kg bw/day (mid-dose)
2800-4400 mg/kg bw/day (high dose)

At 2 week lactation in females there was a slight increase in the sterol levels:
1700-1800 mg/kg bw/day (low dose)
3400-3500 mg/kg bw/day (mid-dose)
8500-9100 mg/kg bw/day (high dose).
Details on mating procedure:
No data
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
2 generations (Approx. 35 weeks)

Frequency of treatment:
Daily
Details on study schedule:
- F1 parental animals not mated until 10 wk after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 6 wk of age.
Remarks:
Doses / Concentrations:
1, 2 and 5 % (made up from 1.6, 3.2 and 8.0% test material; equivalent to 500 – 1300 mg/kg bw/day (low dose), 1000 -2600 mg/kg bw/day (mid-dose) and 2800-4400 mg/kg bw/day (high dose)
Basis:
nominal in diet
No. of animals per sex per dose:
28 rats/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
No data
Positive control:
Not applicable
Parental animals: Observations and examinations:
DAILY CLINICAL OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly (and for mated females on Days 0, 7, 14 and 21 of gestation, and on Days 1, 7, 14 and 21 post partum)

FOOD CONSUMPTION AND COMPOUND INTAKE: Weekly (not during mating)
Food effeciency: Pre-mating food efficiency (weeks 0-5 and 6-10 pre-mating, Days 0-21 gestation and Days 1-14 postpartum)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER: Test substance intake
Oestrous cyclicity (parental animals):
In parent females, 3 weeks post weaning vaginal smears were performed to establish oestrus cycle length.
Sperm parameters (parental animals):
No data
Litter observations:
PARAMETERS EXAMINED
F1 and F2 litters were examined at 1, 4, 7, 14 and 21 d (weaning).
- Litter evaluation: On Days 4, 7, 14 and 21 post partum litters were evaluated for size, sex, still and live births, malformed offspring.
- Pup weight: On Days 1, 4, 7, 14 and 21 post partum

GROSS EXAMINATION OF DEAD PUPS:
- Weanling necropsy and histology of abnormal tissues (stillborns and intercurrent deaths)

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving F0 males were sacrificed 3 weeks post partum
- Female animals: All surviving F0 females were sacrificed 6 weeks post partum

GROSS NECROPSY & HISTOPATHOLOGY
- Necropsy and histology of F0 and F1 parental animals

Postmortem examinations (offspring):
SACRIFICE
- F1 offspring not selected as parental animals: 10 male and 10 female were subject to necropsy and tissue analysis.
Statistics:
No data
Reproductive indices:
Sexual maturation, fertility and reproductive performance were also monitored.
Offspring viability indices:
No data
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
BODY WEIGHT
Males: Mean body weights of F0 and F1 males at all dose groups was consistently lower than in controls. Differences in body weights were up to 6% in F0 and 8.5% in F1 animals. It was statistical significant at some time points in the highest dose group.
These differences were reflected in slight, and sometimes statistically significant differences between high dose males and controls in body weight change and food consumption and efficiency.

Females: In F0 females, there was slight (statistically insignificant) increase in body weight.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption and efficiency: Slight and at some point statistically significant differences between high dose males and controls.

FERTILITY AND REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Fertility and reproductive performance parameters were not significantly altered by sterol
treatment in either generation.

GROSS AND HISTOPATHOLOGYPATHOLOGY (PARENTAL ANIMALS): There were no consistent or dose related effects on organ weights or histopathology in either generation.

Key result
Dose descriptor:
NOAEL
Effect level:
>= 2 800 - < 4 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fertility, reproductive performance and litter data were not significantly altered by sterol treatment in either generation. There were no consistent or dose related effects on organ weights or histopathology in either generation.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
There were also no dose-related changes in litter data.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 2 800 - < 4 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects
Key result
Critical effects observed:
no
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 2 800 - < 4 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

None

Conclusions:
Under the conditions of this study, the NOAEL of the substance in rats was determined to be 5% (equivalent to 2,800-4,400 mg/kg bw/day in males and females) in diet based on lack of effects at the highest tested dose.
Executive summary:

A two generation reproductive toxicity study was conducted in Wistar rats to assess the dietary effects of the constituent plant sterols. 28 Wistar rats/sex/dose were administered dietary concentrations of 0, 1, 2 and 5% plant sterol; equivalent to 0, 500–1,300 mg/kg bw/day (low dose), 1,000-2,600 mg/kg bw/day (mid-dose) and 2,800-4,400 mg/kg bw/day (high dose) of the substance . Treatments began in F0 animals 10 weeks before mating and continued until weaning (3 weeks post parturition) when F0 males were sacrificed. F0 females were maintained for another 3 weeks and then sacrificed. During this entire period, various parameters were examined. Selected pups of F1 generation were treated with the substance in diet at the same levels as their F0 parents for 10 weeks pre-mating. The treatment, observation and sacrifice schedule followed that of the F0 generation. F1 and F2 litters were examined at 1, 4, 7, 14 and 21 d (weaning). Both in the first and second generations, no dose-related clinical observations were recorded. Fertility and reproductive performance parameters were not significantly altered by the substance treatment in either generation. There were also no dose related changes in litter data. There were no consistent or dose related effects on organ weights or histopathology in either generation. Slight significant differences of body weight were observed in males compared to controls. These differences were reflected in slight, and sometimes statistically significant differences between high dose males and controls in body weight change and food consumption and efficiency. Under the conditions of this study, the NOAEL of the substance in rats can be concluded as 5% (equivalent to 2,800-4,400 mg/kg bw/day in males and females) in diet based on lack of effects at the highest tested dose (ANZFA, 2001).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
800 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
168
Species:
rat
Quality of whole database:
all studies with constituents were taken from peer reviewed or regulatory opinions; NOAEL based on an one-generation dietary reproductive toxicity study with d-alpha-tocopheryl poly(ethylene glycol) 10000 succinate also known as tocophersolan
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In absence of reproductive toxicity study with the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ the endpoint has been assessed based on studies for substances representative of the main constituents, which can be categorised as glycerides, fatty acids or fatty acid methyl esters (which will eventually hydrolyse to fatty acids (Mattson and Volpenhein, 1972)) and unsaponifiable matter (including tocopherols, sterols, squalene and hydrocarbons). As a large number of studies have been conducted on the individual constituents, particularly in the context of nutritional research, for practical reasons, only a limited number of studies are reported below:

Glycerides

Dietary exposure to 8.75% (i.e., approximately 4,375 mg/kg bw/day) glycerides with chain lengths of C8 - 18, including C18-unsatd. was assessed in a two-generation reproductive toxicity study in gerbils (Temmermans et al., 1988). No significant effect on frequency of litters, mean litter size, total number of newborns or death in weanling stage were found in the second generation. This is in line with findings from studies conducted with other glycerides having long chain lengths such as C16-18, including C18-unsatd. and C18-unsatd. hydroxy (Irwin, 1992; Manorama et al., 1993; Coquet et al., 1977). Across all studies for glycerides, tested doses ranged from 8.75 to 15% in diet. No significant toxicity was seen at any of the doses tested. The highest oral NOAEL was 15% in diet which is equivalent to 7,000 to 17,000 mg/kg bw/day, from a 13-week combined repeated dose and reproduction/developmental screening (feeding) study.

Fatty acids

A number of studies have been conducted on individual fatty acids, as summarised in HERA (2002). At 15% in diet (ca. 7,500 mg/kg bw/day) for 10-16 weeks, oleic acid (C18) did not affect fertility in male rats but appeared to interfere with parturition and mammary gland development in females. The information is however insufficient to be able to verify and conclude on these results. A three-generation study with an oil containing 76% capric acid (C10) administered via diet did not produce any reproductive effects (Hendrich et al., 1993).

Unsaponifiable matter

Tocopherols

Tocopherols and its derivatives have been tested for reproductive toxicity in a series of studies (Tomassi and Silano, 1986 and Fiume, 2002). No toxicologically significant adverse effects were observed. Further, as per CIR 2018 report, dietary dosing of tocopherol derivatives (tocopheryl succinate up to 75 mg/kg bw/day and tocopherol acetate up to ≤1600 mg/kg bw/day) generally did not have any reproductive or developmental effects in rabbits, hamsters, rats and/or mice. Also, structurally similar D-alpha-tocopheryl poly(ethylene glycol) 10000 succinate also known as tocophersolan was not found to produce adverse effects on fertility and other reproductive parameters in a one-generation dietary reproductive toxicity study at dose levels up to 2% (i.e., equivalent to 800 mg/kg/day in males and 1028 mg/kg bw/day in females) in rats (CIR, 2002, JECFA, 1987). Nevertheless, excess dietary vitamin E (at doses 10000 IU vitamin E/kg), as well as vitamin E deficiency, for a prolonged period of time, can interfere with normal reproductive functions in female rats (Yang, 1977). Alpha-tocopherol (CAS 59-02-9) was found to be inactive in 30 endocrine-related ToxCast assays investigating the EATS modalities (i.e., estrogen (n=12), androgen (n=10), thyroid (n=6) and steroidogenesis (n=3) under the EDSP-21 program.

Sterols:

A 2-generation reproduction study in rats fed up to 5% sterols in diet (ca. 2800-4400 mg/kg bw/day) for 10 weeks prior to mating then throughout gestation, lactation and weaning revealed no significant effects on F0 or F1 generations (ANZFA, 2001). Further, there were couple of studies with beta-sitosterol which revealed adverse effects on estrous cycle and male sperms following dosing through sub-cutaneous route (Tice, 1999). However, these effects have not been reviewed further as the exposure route was not considered relevant under the foreseeable use conditions. Further, beta-sitosterol was found to be inactive in 17 endocrine-related ToxCast assays investigating the EATS modalities (i.e., estrogen (n=6), androgen (n=6), thyroid (n=2) and steroidogenesis (n=3)) under the EDSP-21 program.

Squalene:

Squalene is a common component of the human diet through olive oil and other oils and fats and it is consumed in a quantity between 30 and 200 mg/day, without adverse effect in a whole human life, with no limitation during pregnancy and no effect have ever been reported in normal use on fertility, development or teratogenicity. In addition, squalene has been used since decades in cosmetic formulations, creams and similar, where a daily application is performed during a lifetime by humans. No effects have ever been reported, both about eventual systemic toxicity, fertility or reproduction. For practical reasons some of the available weight of evidence information has been presented below:In a reproductive toxicity study in rats the animals were assigned to four dose groups which received either phosphate buffer saline (PBS), squalene-based adjuvant (AS03) or AS03 adjuvanted Arepanrix H5N1 (i.e. Q-H5N1) influenza vaccine. Doses were intramuscularly administered in the rear limbs 28 days prior to cohabitation (with an untreated male) and on gestation days (GD) 7, 9, 12, and 16 and on postnatal day (PND) 7 (littering cohort dams only). Dams were subject to section on day 21, or to deliver normally and assessments in the latter group were carried out to postnatal day 25. Serological analysis proved vaccine exposure of dams during pregnancy and exposure of foetuses and pups to anti-H5N1 antibodies. There were no effects on any of the parameters evaluated for the F0 generation and all dams survived until their scheduled termination. There were no abnormalities on c-section data or fetal examinations and in the pups followed to postnatal day 25, no toxic effects were observed.

  • In addition, two supportive studies assessed the effect of both Fluarix and FluLaval influenza vaccines, and both AS03 (squalene-based adjuvant) alone and AS03 with H5N1 antigen produced with Fluarix-process, on the embryo-fetal and peri- and post-natal development in naïve or pre-immunised rats following intramuscular administration. In the studies conducted with Fluarix and FluLaval seasonal vaccines, there were no findings in the F0 females or F1 offspring that were considered related to treatment. No signs of maternal toxicity were observed during the reproductive and developmental study performed in rats. Likewise, treatment of naïve or pre-immunized female rats with the AS03-adjuvanted Pandemrix H5N1 or the AS03 adjuvant alone on Days 6, 8, 11 and 15 of gestation did not adversely affect the embryofoetal development or pre- and post-natal development of the offspring. Treatment with the AS03-adjuvanted Pandemrix H5N1 influenza vaccine prior to pairing did not adversely affect the mating performance or fertility of the females (EMEA, 2009).
  • A study was conducted to determine the effects of squalene on reproductive performance in boars. Groups of 6 male and female boars were fed a diet containing 0 (control), 10, 20 or 40 mg/kw bw/day squalene in basal diet for 60 days. Feeding of squalene at 10 mg/kg bw/day neither significantly increase the reproductive performance and serum testosterone levels, nor reduced the levels of leptin in boars. However, feeding with supplemental squalene at 20 or 40 mg/kg bw/day significantly improved the reproductive performance as evidenced by dramatically reduced the time for mating, increased semen volume and motility, and increased the size of litter as compared with that in controls. In addition, higher dose of squalene significantly reduced the levels of serum leptin, accompanied by elevated levels of testosterone, as compared with that in controls. These data clearly indicate that feeding with squalene can improve the reproductive performance in boars (Zhang, 2008).
  • A study was conducted to evaluate the influence of Squalene on radiation-induced biochemical, histological and embryological changes in Sprague Dawley rats. Squalene was orally administered to rats (5 mL/kg bw/day of 2% squalene i.e., equivalent to 72 to 90 mg/kg bw/day) throughout 60 days (43 days before and 17 days following gestation in pregnant rats) before whole body gamma irradiation with 4Gy. In the mated male rats and their pregnant counterparts, Squalene significantly restored the radiation induced male and female sex hormonal abrupt changes especially in female rats. Squalene administration to pregnant rats before irradiation at gestational day 17 improved the fetal survival ability as identified by the disappearance of resorption sites in the tested maternal uteri. Therefore, it could be concluded that Squalene exhibited protecting growing embryos property against radiation induced intrauterine fatal effect (Ibrahim, 2012).

Hydrocarbons:

In a one generation reproductive/developmental toxicity study, rats were exposed to C16–C30 mineral oil (60% paraffins, 40% cycloparaffins) by dermal administration at daily doses of 125, 500, or 2000 mg/kg bw/day. The treatment began 10 weeks prior to mating and continued through a 3-week mating period and continued to gestational day 20, at which point dosing was suspended, and the animals were held without further treatment to postnatal day 21, when all surviving dams and offspring were sacrificed. There were no clinical signs of systemic toxicity and no effects on maternal body weight or food consumption. There were no effects on survival or development of offspring, no evidence of malformation, and there were no pathological findings. The NOAEL for mineral oil in the study was considered >2000 mg/kg bw/day for reproductive performance effects (gonadal function, all 4 stages of the estrus cycle, fertilization, implantation, length of gestation, and parturition) (Mckee, 2015; Dalbey, 2014).

Taken together, the above evidence suggests that the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is not expected to be a reproductive toxicant.

Effects on developmental toxicity

Description of key information

The available weight of evidence from studies conducted on substances representative of the different constituents (glycerides, fatty acids, tocopherols, sterols, squalene and hydrocarbons) indicated that the test substance is not likely to be a developmental toxicant.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1972
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Developmental toxicity/teratogenicity potential was observed in a two generation reproductive toxicity study after dietary administration of the constituent 15% partially hydrogenated soybean oil in Sprague Dawley rats.

Groups of 25 pairs of two generations of male and female rats were fed diets containing 15% of fresh partially hydrogenated soybean oil. F0 generation was exposed to the test material from weaning and F1 generation from conception. The first two litters of each generation were
permitted to be born naturally. During the third pregnancy of each generation, one-half of the females were sacrificed on Day 13 of gestation and inspected for early embryonic death. The remaining females were sacrificed on Day 21 of gestation, and the fetuses were examined for either skeletal or soft tissue abnormalities.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Diet: Semipurified rat diet, ad libitum


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly
- Mixing appropriate amounts with (Type of food): Semipurified rat diet
- Storage temperature of food: Under refrigeration
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Details on mating procedure:
- Proof of pregnancy: Cornified cells and spermatozoa in vaginal smear; referred to as Day 0 of pregnancy
Duration of treatment / exposure:
F0 generation: From weaning and F1 generation: From conception
Frequency of treatment:
Daily ad libitum in diet

Duration of test:
Up to Day 13/21 of gestation or up to weaning of offsprings (see further details on study design for explanation)
Remarks:
Doses / Concentrations:
15%
Basis:
nominal in diet
No. of animals per sex per dose:
25 animals/sex
Control animals:
no
Details on study design:
The first two litters of F0 and F1 generations (i.e. F1a, F1b, F2a and F2b) were permitted to be born naturally. All F1a, F2a and F2b litters were discarded at weaning. F1b rats were allowed to grow for further mating. During the third pregnancy of each generation (i.e. F1c and F2c), one-half of the females were sacrificed on Day 13 of gestation and inspected for early embryonic death. The remaining females were sacrificed on Day 21 of gestation, and the fetuses were examined for either skeletal or soft tissue abnormalities.
Maternal examinations:
BODY WEIGHT: Weekly during the first 8 wks (after weaning)


FOOD CONSUMPTION: Weekly during the first 8 wks (after weaning)


POST-MORTEM EXAMINATIONS: Yes
- Organs examined: Heart, lung, stomach, liver, kidney, adrenals, small and large intestine, spleen, gonads, bladder, pancreas and mesenteric lymph nodes


Ovaries and uterine content:
The uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions: Yes
Fetal examinations:
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
Statistics:
The data were analyzed statistically by the Analysis of Variance and Chi-square method

Indices:
None
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No evidence of maternal toxicity in both F0 and F1 generation
Key result
Dose descriptor:
NOAEL
Effect level:
15 other: % in diet
Basis for effect level:
other: no treatment-related effects
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No dead fetuses or any evidence of teratogenic effects
Key result
Dose descriptor:
NOAEL
Effect level:
15 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects on development of the fetuses
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Table 1. Effect of partially hydrogenated soybean oil on development of rat fetus

 

Generation

F1c

F2c

No. of fetuses examined for soft-tissue defects

65

75

Fetuses with soft-tissue defects

0

0

No. of fetuses examined for skeletal defects

30

37

Fetuses with skeletal defects

0

1

Conclusions:
Under the conditions of this study, the NOAEL for maternal and developmental toxicity was considered to be 15% of the substance in diet.
Executive summary:

A study was conducted to evaluate the developmental toxicity/teratogenicity potential of the constituent 15% ‘glycerides, C8 -18 and C18 -unsatd.’ (as partially hydrogenated soybean oil) in Sprague Dawley rats. Groups of 25 pairs of two generations of male and female rats were fed diets containing 15% of the substance. The F0 generation was exposed to the substance from weaning and the F1 generation from conception. The first two litters of each generation were allowed to be born naturally. During the third pregnancy of each generation, one-half of the females were sacrificed on Day 13 of gestation and inspected for early embryonic death. The remaining females were sacrificed on Day 21 of gestation and fetuses were examined for either skeletal or soft tissue abnormalities. No evidence of any maternal or developmental toxicity (including teratogenicity) was observed in any of the generations. Under the conditions of this study, the NOAEL for maternal and developmental toxicity was considered to be 15% of the substance in diet (Nolen, 1972).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
A two generation reproductive toxicity study was conducted in gerbils to assess the effects of dietary exposure to 8.75% w/w palm kernel oil (PO).
Adult Mongolian gerbils (highly resistant to atherosclerosis) and their sucklings (first generation) were randomly assigned to two groups of 5 couples each (group 1: basal diet; group 2: 8.75% w/w PO in diet). In the second generation parameters such as frequency of litters, number of pups, mean litter size, mean weight, postnatal mortality were observed.
GLP compliance:
not specified
Limit test:
no
Species:
other: gerbil
Strain:
other: Mongolian
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Adult gerbils and their sucklings (F1) were used at the initiation
- Housing: Housed in couples in standard laboratory cages with a wire mesh lid with food hopper. The bedding consisted of wood shavings.
- Diet (e.g. ad libitum): Diets manufactured by Hope Farms BV (Woerden, The Netherlands) were used. The test diet contained increased protein (26%), biotin, choline, inositol and vitamin E (50%) and, cellulose (1%).
- Water (e.g. ad libitum): Water acidified with HCI (pH 3), ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature: 20°C
- Humidity: 50%
- Photoperiod: 10 h light/14 h dark
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
No data
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Not applicable
Details on mating procedure:
No data
Duration of treatment / exposure:
2 generations (specific period not mentioned)
Frequency of treatment:
Daily
Duration of test:
Up to 6 months of F2 generation
Remarks:
Doses / Concentrations:
8.75% w/w
Basis:
nominal in diet
No. of animals per sex per dose:
5 couples per group
Control animals:
yes, plain diet
Details on study design:
No data
Maternal examinations:
No data
Ovaries and uterine content:
Not examined
Fetal examinations:
The following parameters were examined in [F2] offspring: Frequency of litters, number of pups, mean litter size, mean weight at 6 months of age and postnatal mortality

Statistics:
The Student-t test for unpaired samples was used to compare the variables of the different groups. For large differences in variance between the samples, the Wilcoxon test for unpaired samples was used. p < 0.05 was considered significant.
Indices:
Litter frequency: calculated by dividing the total number of weeks during which the couples of gerbils within one dietary group were able to breed by the number of litters
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no data

Details on maternal toxic effects:
Not applicable
Key result
Dose descriptor:
NOAEC
Effect level:
> 8.75 other: % w/w
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:not examined

Details on embryotoxic / teratogenic effects:
Not applicable
Key result
Dose descriptor:
NOAEL
Effect level:
8.75 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects on development
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

OFFSPRING PARAMETERS:

 

BODY WEIGHT (OFFSPRING): No significant difference compared to controls was observed (F1 and F2)

FREQUENCY OF LITTERS (F2): No significant effects were observed.


NUMBER OF PUPS (F2): No significant difference was observed (PO: 116 and basal diet (control): 146).


MEAN LITTER SIZE (F2): No significant difference was observed (PO: 4.7±1.7 and basal diet (control): 4.9±1.6).


POSTNATAL MORTALITY (F2): No significant mortality was observed in comparison to basal diet group ( PO: 4% and basal diet (control): 3%)
Conclusions:
Based on the test conditions, the NOAEL for developmental effects in rats was determined to be 8.57% w/w.
Executive summary:

A two generation reproductive toxicity study was conducted in Mongolian gerbils to assess the effect of the constituent ‘glycerides, C8 -18 and C18 -unsatd.’ (as palm kernel oil). Adult Mongolian gerbils (highly resistant to atherosclerosis) and their sucklings (first generation) were randomly assigned to two groups of 5 couples each (group 1: basal diet; group 2: 8.75% w/w of the substance in diet). In the second generation parameters such as frequency of litters, number of pups, mean litter size, mean weight, postnatal mortality were observed. No significant effect on frequency of litters, mean litter size, total no. of newborns, mean body weight and no postnatal mortality were found in the second generation of the substance-treated group compared to the basal dietary group. Based on the test conditions, the NOAEL for developmental effects in rats was determined to be 8.57% w/w (Temmerman, 1988).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
A developmental toxicity study was conducted in Wistar rats to assess the effect of the constituent alpha-tocopherol acetate.
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Vehicle:
corn oil
Analytical verification of doses or concentrations:
not specified
Details on mating procedure:
Not reported
Duration of treatment / exposure:
GD 6-15
Frequency of treatment:
Daily
Duration of test:
GD 6-20
Remarks:
Doses / Concentrations:
16, 74.3, 345 or 1,600 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
21, 23, 21, and 22 gravid albino Wistar rats for 16, 74.3, 345 or 1,600 mg/kg bw/day
Control animals:
yes, sham-exposed
Key result
Dose descriptor:
NOAEL
Effect level:
1 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
1 600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects on fetal development
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

None

Conclusions:
Under the test conditions, the NOAEL of the substance for developmental toxicity was determined to be 1,600 mg/kg bw/day.
Executive summary:

A study was conducted to evaluate the developmental toxicity potential of the constituent alpha-tocopherol acetate in Wistar rats. Groups of 21, 23, 21, and 22 gravid albino Wistar rats were dosed orally at 16, 74.3, 345, or 1,600 mg/kg bw/day of the substance in corn oil during Days 6 to 15 of gestation. Body weights were recorded on Days 0, 6, 11, 15, and 20 of gestation. The animals were killed on Day 20 of gestation and the fetuses were examined. The administration of up to 1600 mg/kg bw/day of the substance to pregnant rats had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls. Under the test conditions, the NOAEL of the substance for developmental toxicity was determined to be 1,600 mg/kg bw/day (Fiume, 2002).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Reason / purpose for cross-reference:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
The teratogenic effects of the constituent d-alpha-tocopherol (polyethylene glycol) 1000 succinate (TPGS) were investigated upon dietary exposure to 0, 0.002, 0.2 and 2% (i.e. 1.02, 103 and 1,028 mg/kg bw/day, assuming 0.35 kg as average weight of adult rats and 18 g as daily food consumption) on Days 6 to 16 of gestation. On Day 20 of gestation, the dams were sacrificed, the uteri excised, and the number of implantation sites (live fetuses, dead fetuses, or resorption sites) were counted. All the fetuses were examined for gross anomalies; half were examined for soft-tissue abnormalities (Wilson technique) and half were examined for skeletal defects.
GLP compliance:
not specified
Species:
rat
Strain:
other: Charles River CD
Details on test animals or test system and environmental conditions:
Not reported in the JECFA evaluation report
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
Not reported in the JECFA evaluation report
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Not applicable
Details on mating procedure:
Not reported in the JECFA evaluation report
Duration of treatment / exposure:
GD 6-16
Frequency of treatment:
Daily
Duration of test:
up to GD 20
Remarks:
Doses / Concentrations:
0, 0.002, 0.2, and 2% (i.e., approx. 1, 103 and 1,028 mg/kg bw/day)
Basis:
nominal in diet
No. of animals per sex per dose:
15 pregnant females/dose
Details on study design:
Not reported in the JECFA evaluation report
Maternal examinations:
Not reported in the JECFA evaluation report
Ovaries and uterine content:
On Day 20 of gestation, the dams were sacrificed, the uteri excised, and the number of implantation sites (live fetuses, dead fetuses, or resorption sites) were counted.
Fetal examinations:
All the fetuses were examined for gross anomalies; half were examined for soft-tissue abnormalities (Wilson technique) and half were examined for skeletal defects (stained using alizarin red).
Statistics:
Not reported in the JECFA evaluation report
Indices:
Not reported in the JECFA evaluation report
Historical control data:
Not reported in the JECFA evaluation report
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
Not reported in the JECFA evaluation report
Key result
Dose descriptor:
NOEL
Effect level:
> 1 028 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects: no effects

Details on embryotoxic / teratogenic effects:
No differences were observed between controls and any of the treatment groups with respect to the parameters studied.
Key result
Dose descriptor:
NOAEL
Effect level:
1 028 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

None

Conclusions:
Under the conditions of the study, the NOEL of the substance for teratogenic and fetotoxic effects was determined to be >1,028 mg/kg bw/day, based on lack of developmental effects at the highest tested dose.
Executive summary:

The teratogenic/fetotoxic effects on dietary exposure to d-alpha-tocopherol (polyethylene glycol) 1000 succinate (TPGS) was determined in Charles River CD rats. Groups of 15 pregnant Charles River CD rats were administered the substance through diet at concentrations of 0, 0.002, 0.2, or 2% (i.e. approx. 1.02, 103 and 1,028 mg/kg bw/day) on Days 6 to 16 of gestation. On Day 20 of gestation, the dams were sacrificed, the uteri excised, and the number of implantation sites (live fetuses, dead fetuses, or resorption sites) were counted. All the fetuses were examined for gross anomalies; half of the fetuses were examined for soft-tissue abnormalities and remaining half were examined for skeletal defects. No differences were observed between controls and any of the treatment groups with respect to the parameters studied. Under the conditions of the study, the NOEL of the substance for teratogenic and fetotoxic effects was determined to be >1,028 mg/kg bw/day, based on lack of effects at the highest tested dose (JECFA, 1987).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Secondary literature source with limited details, but used in the Australia New Zealand Food Authority (ANZFA) assessment of phytosterol esters, and followed GLP
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
A two generation reproductive toxicity study was conducted in Wistar rats to assess the dietary effects of 0, 1, 2 and 5% plant sterol (made up from 0, 1.6, 3.2 and 8.0%; equivalent to 0, 500–1300 mg/kg bw/day (low dose), 1000-2600 mg/kg bw/day (mid-dose) and 2800-4400 mg/kg bw/day (high dose). Treatments began in F0 animals 10 weeks before mating and continued until weaning (3 weeks post parturition) when F0 males were sacrificed. F0 females were maintained for another 3 weeks and then sacrificed. During this entire period, various parameters were examined. Selected pups of F1 generation were treated with plant sterols in diet at the same levels as their F0 parents for 10 weeks pre-mating. The F1 and F2 litters were examined at weaning days 1, 4, 7, 14 and 21 for various parameters including developmental toxicity.
GLP compliance:
yes
Remarks:
Lab name not reported in the ANZFA risk analysis report
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld

Route of administration:
oral: feed
Vehicle:
not specified
Details on exposure:
Test substance intake was monitored:
At pre-mating and gestation sterol doses in males and females of F0 and F1 generation were in the ranges:
500-1,300 mg/kg bw/day (low dose)
1,000-2,600 mg/kg bw/day (mid-dose)
2,800-4,400 mg/kg bw/day (high dose)

At 2 week lactation in females there was a slight increase in the sterol levels:
1,700-1,800 mg/kg bw/day (low dose)
3,400-3,500 mg/kg bw/day (mid-dose)
8,500-9,100 mg/kg bw/day (high dose).
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
No data

Duration of treatment / exposure:
2 generations
Frequency of treatment:
Daily
Duration of test:
Approx. 35 weeks
Remarks:
Doses / Concentrations:
1, 2 and 5 % (made up from 1.6, 3.2 and 8.0% test material; equivalent to 500 – 1,300 mg/kg bw/day (low dose), 1,000 -2,600 mg/kg bw/day (mid-dose) and 2,800-4,400 mg/kg bw/day (high dose)
Basis:
nominal in diet
No. of animals per sex per dose:
28 rats/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
No data
Maternal examinations:
DAILY CLINICAL OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly (and for mated females on Days 0, 7, 14 and 21 of gestation and on Days 1, 7, 14 and 21 post partum)

FOOD CONSUMPTION AND COMPOUND INTAKE: Weekly (not during mating)
Food effeciency: Pre-mating food efficiency (weeks 0-5 and 6-10 pre-mating, Days 0-21 gestation and 1-14 postpartum)

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OTHER: Test substance intake
Ovaries and uterine content:
No data
Fetal examinations:
PARAMETERS EXAMINED
F1 and F2 litters were examined at weaning days 1, 4, 7, 14 and 21
- Litter evaluation: On Day 4, 7, 14 and 21 post partum litters were evaluated for size, sex, still and live births, malformed offspring.
- Pup weight: On Day 1, 4, 7, 14 and 21 post partum

GROSS EXAMINATION OF DEAD PUPS:
- Weanling necropsy and histology of abnormal tissues (stillborns and intercurrent deaths)
Statistics:
No data
Indices:
No data
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
In F0 females, there was slight (statistically insignificant) increase in body weight.

Key result
Dose descriptor:
NOEL
Effect level:
>= 2 800 - < 4 400 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: no treatment-related effects
Key result
Abnormalities:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 2 800 - < 4 400 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects on development of F0 and F1 fetuses
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

None

Conclusions:
Under the conditions of this study, the NOEL of the substance for developmental toxicity in rats was determined to be 5% (equivalent to 2,800-4,400 mg/kg bw/day in males and females) in diet, based on lack of any morphological or pathological findings in pups of F0 and F1 generations.
Executive summary:

A two-generation reproductive toxicity study was conducted in Wistar rats to assess the effect of the constituent plant sterols and was also used to assess any developmental abnormality in pups. In the study, 28 Wistar rats/sex/dose were administered dietary concentrations of 0, 1, 2 and 5% plant sterol (made up from 0, 1.6, 3.2 and 8.0%; equivalent to 0, 500–1,300 mg/kg bw/day (low dose), 1,000-2,600 mg/kg bw/day (mid-dose) and 2,800 -4,400 mg/kg bw/day (high dose) of the substance . Treatments began in F0 animals 10 weeks before mating and continued until weaning (3 weeks post parturition) when F0 males were sacrificed. F0 females were maintained for another 3 weeks and then sacrificed. During this entire period, various parameters were examined. Selected pups of F1 generation were treated with the substance in diet at the same levels as their F0 parents for 10 weeks pre-mating. The treatment, observation and sacrifice schedule followed that of the F0 generation. The F1 and F2 litters were examined at weaning days 1, 4, 7, 14 and 21 for various parameters including developmental toxicity. Both in the first and second generations, no dose-related significant morphological or pathological findings were observed in pups of F0 and F1 generation with the substance at 5% of diet.  Under the conditions of this study, the NOEL of the substance for developmental toxicity in rats was determined to be 5.0% (equivalent to 2800 -4400 mg/kg bw/day in males and females) in diet, based on lack of any morphological or pathological findings in pups of F0 and F1 generations (ANZFA, 2001).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
All studies with constituents were taken from peer reviewed or regulatory opinions; NOAEL based on a development toxicity study with C16-C20, n-alkanes, isoalkanes, cyclic, and <2% aromatics in rats;
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Additional information

In the absence of a development toxicity study with the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ the endpoint has been assessed based on studies for substances representative of the main constituents, which can be categorised as glycerides, fatty acids or fatty acid methyl esters (which will eventually hydrolyse to fatty acids (Mattson and Volpenhein, 1972)) and unsaponifiable matter (including tocopherols, sterols, squalene and hydrocarbons). Asa large number of studies have been conducted on the individual constituents, particularly in the context of nutritional research, for practical reasons, only a limited number of studies are reported below:

Glycerides:

Dietary exposure to 8.75% in diet (ca.4375 mg/kg bw/day) glycerides with chain lengths of C8-18, including C18-unsatd. was assessed in a two-generation reproductive toxicity study in gerbils (Temmermans et al., 1988). No significant effect on frequency of litters, mean litter size, total no. of newborns and death during weanling stage were observed in the second generation. These results are in line with additional evidence of absence of developmental effects obtained from another two-generation study conducted in rats exposed to glycerides of chain length ranging from C16-18, including C18-unsatd (Nolen, 1972). Across all studies, tested doses ranged from 8.75 to 15% in diet. No significant toxicity was seen at any of the tested doses. The highest oral NOAEL could be considered to be 15% in diet, which is equivalent to an estimated 7,500 mg/kg bw/day.

Fatty acids:

No specific information could be found for fatty acids. Several fatty acids (stearic acid, oleic acid and sodium palmitate) are Generally Recognised as Safe (GRAS). Also, fatty acids as a group are permitted as direct food additives (HERA, 2002). Overall, fatty acids are not expected to show any developmental toxicity.

Unsaponifiable matter:

Tocopherols:

The developmental toxicity was evaluated on the basis of several oral pre-natal developmental toxicity studies conducted with structurally similar tocopheryl acetate in rats, mice, rabbits and hamsters. These studies did not reveal any adverse effects up to the highest tested dose of 1,600 mg/kg/day (Fiume (CIR), 2002).

Sterols/sterol esters:

No developmental studies were submitted for evaluation. However, the 2-generation reproduction study with5% sterols in diet (ca. 2800-4400 mg/kg bw/day) revealed no significant morphological or pathological findings in pups of F0 and F1 matings (ANZFA, 2001).

Squalene:

Apart from the epidemiological evidence deriving from the presence of squalene as a component of common edible oils and fat and cosmetic application, several studies have been performed in the framework of the use of squalene as adjuvant in recently developed influenza vaccines.

  • Reproductive and development toxicity study with squalene-based adjuvanted vaccine AFLUNOV in rabbits following intramuscular injections at twice the clinical dose for humans (7.5 µg) before mating and during gestation, did not induce maternal/embryofoetal toxicity or teratogenic and post-natal development effects (EMA, 2010a).
  • Two studies using rabbits investigated the effects of M59-adjuvanated vaccination on either Day 6 through to Day 28 of gestation, or three times before mating and at days 7 and 20 of gestation. Information on the timing of dosing in the rat study was not described. In these studies, MF59-adjuvanted vaccines were well-tolerated, did not cause maternal or embryo–fetal toxicity, were not teratogenic and had no effects on post-natal development. It was concluded that there are no specific concerns for the use of this adjuvanted vaccine in pregnancy. The AS03 adjuvant is present in two pandemic influenza vaccines, Pandemrix® (GSK Biologicals) and Arepanrix® (GSK Biologicals). The potential reproductive toxicity of this adjuvant was tested in rats, with and without influenza antigen. For both products, animals were dosed once before mating and at Days 6, 8, 11 and 15 of gestation for Pandemrix and at Days 7, 9, 12 and 16 for Arepanrix. The only potential treatment-related effect was a somewhat delayed air-righting reflex for some of the offspring of AS03-treated dams in the Pandemrix study. Another pandemic influenza vaccine, Humenza® (Sanofi Pasteur, Lyon, France) contains the adjuvant AF03. Two studies in rats and rabbits have been reported using the adjuvant alone. Two rabbit studies using the AF03-adjuvanted A/California/07/2009 H1N1 influenza vaccine have been conducted with a similar schedule as for Pandemrix (Days -21, -7, 6, 8, 11 and 27) and no embryo–fetal toxicity effects have been noted (Herberts, 2015).
  • In a prenatal developmental toxicity study, six groups of 48 female rats were given a single intramuscular dose on Day -30 and paired with males and 44 rats with a positive indication of mating were treated on Days 6, 8, 11 and 15 after mating. The six groups were: (1) saline, 200 μL; (2) AS03 adjuvant, 200 μL; (3) saline / split H5N1/AS03, 200 μL; (4) split H5N1 / AS03, 200 μL; (5) saline /whole H5N1/A1, 100 μL; (6) whole H5N1/A1, 100 μL. After dosing, 22 were killed at Day 20 and 22 were allowed to deliver and rear their young to Day 25 of age. Measures of reproductive toxicity and maternal health were assessed. There was one unexpected death in a maternal rat: however, this was judged unrelated to the vaccine. Treatment of maternal rats did not adversely affect their clinical condition, body weight or food consumption throughout the study. Mating performance, fertility of maternal rats, and length of gestation or ability to give birth to a live litter were unaffected. Embryo-foetal survival, growth and development were not affected by vaccination. In neonates, the reflex development was unimpaired, but among offspring from dams treated with AS03 13 offspring from 7 litters did not show the air righting reflect before day 21 of age and this effect may be related to treatment. However, AS03 did not affect the attainment of the surface righting reflex or the ability of the offspring to show startle response reflexes or the pupil reflex. No abnormalities were evident on macro pathological examination of the offspring (EMEA, 2008).
  • A large clinical study using primary data collection found that MF59 adjuvanted A/H1N1 influenza vaccine did not result in an increased risk of adverse perinatal events and suggested a lower risk among vaccinated women. These findings should contribute to inform stakeholders and decision makers on the prescription of vaccination against influenza A/H1N1 in pregnant women (Rubinstein, 2013).
  • A retrospective analysis study was conducted to review the nonclinical and clinical data on pregnancy outcomes associated with exposure to MF59®, an adjuvant used in licensed H1N1 pandemic vaccines. MF59 adjuvant is an oil-in-water emulsion, comprising squalene oil in the final dose [9.75 mg] stabilized by the water-soluble surfactant Tween 80 [1.17 mg] and the oil soluble surfactant Span 85 [1.17 mg] in low ionic strength citrate pH 6.5 buffer. To evaluate the reproductive and developmental toxicity effects, non-clinical study data in rats (0.5 mL dose) and rabbits (0.063 mL to 0.125 mL dose) as well as clinical study data in pregnant human female (0.25 mL dose) were evaluated. Also, clinical data analysis of 23,334 subjects receiving MF59-containing influenza vaccines and 40,285 subjects receiving unadjuvanted influenza vaccines and pregnancy exposures during the periconception time window of −30 to +45 days from last menstrual period (LMP) was conducted. There were no adjuvant-related findings observed in any reproductive and developmental toxicity study in the nonclinical assessment. MF59 adjuvant alone or in combination with the egg-derived H5N1 antigen was not maternally toxic, fetotoxic or teratogenic. Furthermore, there were no observed effects on developmental parameters in offspring. A total of 43 pregnancies in recipients of MF59-adjuvanted influenza vaccines and 60 in recipients of unadjuvanted influenza vaccines were identified in the clinical database with similar mean and median age of women in both groups. Overall, the proportion of pregnancy outcomes that were reported as normal – 70% and 75%, respectively – and distribution of outcomes (normal, abnormal and terminated in abortion) were similar in the two groups who had been exposed to MF59-adjuvanted and unadjuvanted influenza vaccines. Similar results were seen when the analysis was focused on exposures occurring within the interval of −30 to +45 days of the LMP; the distribution by pregnancy outcomes in the MF59-adjuvanted and unadjuvanted influenza vaccine groups was similar and the proportion with normal outcomes were 61% and 68%, respectively. Under the study conditions, the nonclinical and clinical data analysis on pregnancy outcomes associated with exposure to MF59®, an adjuvant (squalene with emulsifying agents Tween 80 and Span 85) used in licensed H1N1 pandemic vaccines demonstrated no evidence of teratogenicity or impact on fetal or early perinatal development in animals and human population (Tsai, 2010).

Hydrocarbons:

Developmental toxicity studies were conducted with C16–C30 mineral oil (60% paraffins, 40% cycloparaffins) in Sprague-Dawley rats following exposure during gestational days 6-19 via oral (5000 mg/kg bw/day), dermal (2000 mg/kg bw/day), or inhalation (1000 mg/m3 as aerosol, 6 hours/day). The dams were sacrificed on gestational day 20, and the uterine contents were examined. There was no evidence of maternal toxicity or fetal effects. Therefore, the NOAEL/NOAEC considered were the highest doses/concentrations used in this study (Mckee, 2015; Dalbey, 2014).

Development toxicity study with C16-C20, n-alkanes, isoalkanes, cyclic, and <2% aromatics in rats also did not reveal signs of maternal toxicity or treatment-related adverse effects on fetal development up to 1000 mg/kg bw/day (highest dose). The CIR panel also concluded that no significant toxicity was identified in hydrocarbons oral or inhalation exposure studies for reproductive and developmental toxicity end point (CIR, 2012).

Taken together, the above evidence suggests that the test substance, ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ is not expected to be a developmental toxicant.

Justification for classification or non-classification

Based on the available weight of evidence information on the constituents, the test substance ‘squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation’ does not warrant classification for reproductive and development toxicity according to EU CLP criteria (EC 1272/2008).

Additional information