Squalene-rich fraction obtained from vegetable oil deodorizer distillate by transesterification, crystallisation and vacuum distillation

Toxicity to reproduction

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Remarks:

FDA Good Laboratory Practices Regulations (21 CFR 58)

Species:

mouse

Strain:

B6C3F1

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: Individually caged 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: 15 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

Details on route of administration:

0, 0.62, 1.25, 2.5, 5.0 or 10% in diet

Vehicle:

unchanged (no vehicle)

Details on oral 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.
- Mixing appropriate amounts with (Type of food): 10% (100 mg/g) determined by gravimetric analysis, and blends at 0.5% (5 mg/g) determined by HPLC analysis.
- 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.

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

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 mice per sex per dose

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed initially and 1 x wk thereafter


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


OTHER: ORGAN WEIGHTS:
- Organ weights were determined to the nearest milligram for the liver, right kidney, right testicle, heart, thymus, and lungs. All tissues were preserved in 10% neutral buffered formalin.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
- Necropsy and Histological Examinations: Complete histopathology examinations were conducted on all mice from the control and 10% dose groups. The following tissues were routinely processed for preparation of histological sections and microscopic examination:
adrenal glands, brain, cecum, colon, duodenum, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, esophagus, eyes (if grossly abnormal), femur (including marrow), heart, ileum, jejunum, kidneys, liver, lungs and main stem bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral glands, rectum, salivary glands, skin, spinal cord and sciatic nerve (if neurologic signs present), spleen, forestomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions and tissue masses including regional lymph nodes.

Other examinations:

REPRODUCTIVE TOXICITY SCREEN:
- Parameters examined: Sperm motility and morphology were evaluated at necropsy and vaginal cytology
- Time schedule for examinations: Sperm motility and morphology: At necropsy; Vaginal cytology: At 12 wk

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)

Clinical signs:

no effects observed

Mortality:

no mortality 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

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Details on results:

MORTALITY: No effect on survival up to 10% concentration of castor oil in diet.


BODY WEIGHT AND WEIGHT GAIN: No significant effects were observed.
- However the results observed were: Mean body weights of exposed male mice generally were lower than controls, while mean body weights of exposed females generally were higher. These differences were not related to dietary concentrations of castor oil, except that mean body weights of male mice receiving the 10% castor oil diet were consistently lower than those of control mice from week 3 through the end of the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No statistically significant differences in average food consumption were observed, although food consumption by female mice receiving diets containing 10% castor oil was slightly lower than controls.


ORGAN WEIGHTS:
- Liver weights were increased in male and female mice at both 5% or 10% of castor oil.
- Kidney weights were increased in female mice at both 5 % and 10 % of castor oil.


GROSS PATHOLOGY: No morphologic changes were observed


HISTOPATHOLOGY: NON-NEOPLASTIC: No compound-related lesions were observed in any organ or tissue of mice exposed to castor oil in the diet.


OTHER FINDINGS: REPRODUCTIVE TOXICITY SCREEN: No adverse effects were observed in any male (testes weight, epididymal sperm motility, density, or testicular spermatid head count) or female mice (estrual cycle length, or time spent in each phase of the cycle) reproductive parameters.

Dose descriptor:

NOAEL

Effect level:

10 other: % in diet (i.e. ca. 14,600 - 20,000 mg/kg bw)

Sex:

male/female

Basis for effect level:

other: no treatment-related effects observed

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of this study, the NOAEL of the substance in mice was determined to be 10% in diet (i.e. ca. 14,600 - 20,000 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 male and female B6C3F1 mice in order to evaluate the sub-chronic and reproductive toxicity of the constituent castor oil. Mice were exposed for 13 weeks to castor oil at 0, 0.62, 1.25, 2.5, 5.0 or 10% in the diet. Mortality, bodyweight and food consumption were recorded throughout the study. Organ weights were determined, and gross pathology as well as histopathology conducted at termination. Sperm motility, morphology and vaginal cytology were evaluated at various time intervals. Exposure to castor oil at dietary concentrations as high as 10% did not affect survival or body weight gains. Liver weights were increased in male and female mice as of 5% castor oil in the diet. However, there were no histopathological lesions associated with these liver changes, nor were there any compound-related morphologic changes in any organ. 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 the female estrous cycles. Thus, no significant adverse effects of castor oil were noted. Under the conditions of this study, the NOAEL of the substance in mice was determied to be 10% in diet (i.e. ca. 16000 mg/kg bw/day based on actual feed consumption and body weight data) (Irwin, 1992).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Glycerides, C16 and C18-unsatd. and C18-unsatd. hydroxy

IUPAC Name:

Glycerides, C16 and C18-unsatd. and C18-unsatd. hydroxy

Details on test material:

- Name of test material (as cited in study report): Castor oil (CAS N° 8001-79-4, EC N° 232-293-8). Under the SDA nomenclature, the name of this substance is ‘Glycerides, C16 and C18-unsatd. and C18-unsatd. hydroxy'
- Physical state: Liquid
- Analytical purity: Purity and identity analyses were conducted by Midwest Research Institute (MRI) (Kansas City, MO)
- Analytical method used: Karl Fischer water analysis, thin layer and high performance liquid chromatography, and a battery of U.S. Pharmacopeia (USP) standard analyses for castor oil
- Composition of test material, percentage of components:
- Lot/batch No.: #L-5G30-01
- Other: Source: Cas Chemical, Inc.

Test animals

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

Administration / exposure

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

Doses / concentrations

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

Details on results (F1)

Not applicable

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

None

Applicant's summary and conclusion

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).