Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From Apr. 1988 to Jul. 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to accepted guidelines in compliance with GLP.
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
The core study was conducted with groups of 10 rats and 10 mice per sex, each group receiving diets containing castor oil at various doses, continuously for 13 wk. Ten additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters.

At the study termination, all core-study animals were euthanized by CO2 anesthesia, and complete necropsies were performed.

To screen for potential reproductive toxicity, sperm motility and morphology were evaluated at necropsy, and vaginal cytology was evaluated on core-study animals during the wk just preceding necropsy, following published procedures (Morrissey et al., 1988). For the 12 d prior to termination, females were subject to vaginal lavage with saline. The relative preponderance of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were used to identify the stages of the estrual cycle.

Sperm motility was evaluated at necropsy and was presented as spermatid heads per total testis and per gram of testis.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Castor oil
- Analytical purity: purity and analyses were conducted by Midwest Research Institute (MRI) (Kansas City, MO). MRI reports on the analyses performed in support of the castor oil studies are on file at the National Institute of Environmental Health Sciences (research triangle park, NC)
- Composition of test material, percentage of components: 87% ricinoleic, 7% oleic, 3% linoleic, 2% palmitic, 1% stearic and trace amounts of dihydroxystearic.
- Lot/batch No.: L-5G30-01
- Stability under test conditions: Monitored by determination of peroxide content and by HPLC. No deterioration of the castor oil study material was observed over the course of the study.
- Other: Obtained from Cas Chemical Inc. (Bayonne, NJ)

Test animals

Species:
other: rat and mouse
Strain:
other: F344/N rats and B6C3F1 mice
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA)
- Age at study initiation: 6 wk
- Weight at study initiation:
Males (rat): 126-132 g
Females (rat): 107-110 g
Males (mouse): 22.6-23.0 g
Female (mouse): 17.2-17.7 g
- Fasting period before study: No
- Housing:
Rat: The rats were housed 5 per cage. Polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets were used.
Mouse: The mice were housed individually. Polycarbonate cages lined with heat-treated hardwood chips and covered with polyester filter sheets were used.
- Diet (e.g. ad libitum): Ad libitum; feeders were changed twice per wk throughout the study
- Water (e.g. ad libitum): The cages were stored on stainless steel racks equipped with an automatic watering system.
- Acclimation period:
Rat: 14 d
Mouse: 15 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 (68-76°F)
- Humidity (%):42-72 %
- Air changes (per hr): 10 times/h. Incoming air was filtered to remove particulates
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE, AGE: From: 16 wk To: 19 wk

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): 2/wk
Details on mating procedure:
Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Periodic analysis of the castor oil-formulated diets was conducted by HPLC at the study and analytical chemistry laboratories. Three complete sets of formulated diet mixtures were analyzed. All but a single sample were within specifications (±10% of the target concentration). A single low-dose mixture which did not meet specifications was remixed and found to be within specifications before it was given to the animals. 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
Frequency of treatment:
Daily
Details on study schedule:
Not applicable
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
Control animals:
yes, concurrent no treatment
Details on study design:
None
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

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
Oestrous cyclicity (parental animals):
The relative preponderance of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were used to identify the stages of the estrual cycle.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
- Sperm morphology.
- Sperm motility which was evaluated at necropsy as follows: The left epididymis was removed and weighed; the cauda epididymis was removed at the junction of the vas deferens and the corpus epididymis, then weighed. The sperm that were removed from the epididymis were dispersed and the number of moving and non-moving sperms were counted in 5 fields of 30 sperm or less on each slide. Sperm density was then determined using a hemocytometer.

Homogenization spermatid nuclei were enumerated using a hemocytometer; the data were expressed as spermatid heads per total testis and per gram of testis.
Postmortem examinations (parental animals):
GROSS NECROPSY
- A complete necropsy was conducted

HISTOPATHOLOGY / ORGAN WEIGHTS
Organ weights: Liver, right kidney, right testicle, heart, thymus, and lungs

The following tissues were routinely processed for preparation of histologic 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 mainstem 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, fore-stomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions and tissue masses including regional lymph nodes. A complete histopathologic examination was conducted on all rats and mice from the control and the 9.67 ± 0.54 mg/L dose groups. Liver was examined from male rats in all other dose groups, and histologic sections of gross lesions were examined from all rats.
Statistics:
Body weight and organ weight data were statistically analyzed within each sex by one-way analysis of variance tests, followed by Dunnett's t-test if pair-wise comparisons were indicated (p < 0.05)(Dunnett, 1955).

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
Rat: No clinical signs or mortality were observed in the groupe tested at any dose level.

Mouse: No clinical signs or mortality were observed in the group tested at any dose level.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
- Rat, male: Mean body weights of rats receiving diets containing castor oil did not differ significantly from controls

- Rat, female: Mean body weights of exposed female rats were slightly lower than the mean body weights of controls but the differences were not dose-related.

- Mouse, male: There were no statistically significant differences in average food consumption among groups. The mean body weights of exposed male mice generally were lower than controls.

- Mouse, female: Female mice receiving diets containing 9.67 mg/L bw was slightly lower than controls.
Mean body weights of exposed females generally were higher than the controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS):
Rat: No effects observed at any dose level

Mouse: No effects observed at any dose level

REPRODUCTIVE FUNCTION: (PARENTAL ANIMALS):
- Rat, male: 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. There was some variation in epididymal weights, however, their small magnitude and the absence of changes in other endpoints suggested that there was little or no evidence of any reproductive toxicity associated with castor oil exposure.

- Mouse, male: No was no adverse effects on any male at any dose level. The low value for sperm motility in control mice was attributed to poor preparative technique.

ORGAN WEIGHTS (PARENTAL ANIMALS):
- Rat, male: Absolute liver weights and the liver-to-body-weight ratio were increased in male rats that received diets containing the highest dose of castor oil. Heart-to-body-weight ratios were increased in groups of male rats receiving 0.62, 2.64 and 9.67 mg/L bw diets; however, absolute heart weights were not increased, and the differences in body weight ratios were small and not considered treatment related. Using light microscopy, it was determined that there were no morphologic changes associated with the slight differences in organ weights between groups.

- Rat, female: No effects reported.

- Mouse, male: Liver weights were increased in male mice that received diets containing 4.91 or 9.67 mg/L castor oil.

- Mouse, female: Liver weights were increased in female mice that received diets containing 4.91 or 9.67 mg/L castor oil. Kidney weights were increased in female mice that received 4.91 or 9.67 mg/L diets. Using light microscopy, it was determined that there were no morphologic changes associated with the slight differences between groups in organ weights.


HISTOPATHOLOGY (PARENTAL ANIMALS):

- Rat: Histopathologic examination revealed an absence of compound-related lesions in any organ or tissue of rats exposed to castor oil in the diet.

- Mouse: Histopathologic examination revealed an absence of compound-related lesions in any organs or tissues of mice exposed to castor oil in the diet.

OTHER FINDINGS (PARENTAL ANIMALS):
- Rat: Hematological effects of the castor oil diets among male rats included a slight decrease in MCHC at Day 21 in those receiving the 9.67 mg/L diet; a statistically significant decrease in MCV among the 9.67 mg/L group; a decrease in MCH among the 4.91 mg/L and 9.67 mg/L groups; and an increase in platelets among the 1.26 mg/L, 4.91 mg/L, and 9.67 mg/L groups. The only change observed among female rats was a statistically significant decrease in reticulocyte counts at Day 5 in groups receiving the 0.62 mg/L or 9.67 mg/L diets. None of these changes was considered biologically significant. A treatment- and dose-related increase in the activity of serum alkaline phosphatase was observed in male and female rats at Day 5 and 21, and at study termination. Total bile acids
16NTP TOX 12, castor oil were increased among males receiving the higher dietary levels at Day 5 and 21 but were not increased at study termination. Other minor changes included increases in albumin observed at study termination in males receiving 4.91 mg/L diets and at Day 5 in females receiving 9.67 mg/L diets, and an increase in urea nitrogen at study termination in males that received 0.62 mg/L diets and a decrease at Day 5 in females that received castor oil at 9.67 mg/L in the diet.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
>= 9.13 - <= 10.21 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Rat. Effect level based on reproductive parameters
Dose descriptor:
NOAEC
Effect level:
>= 9.13 - <= 10.21 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mouse. Effect level based on reproductive parameters

Results: F1 generation

Details on results (F1)

Not applicable

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Under the test conditions, a reproductive NOAEC value for male/female rats and mice was determined to be 10% (i.e., ca. 5,000 mg/kg bw/day in rats and 13,000 mg/kg bw/day in mice), after 13 weeks of exposure to castor oil.
Executive summary:

A 13 week study was conducted according to a method equivalent/similar to OECD Guideline 422 to evaluate the reproductive toxicity effects on mice and rats fed with castor oil.

The core study was conducted with groups of 10 rats and 10 mice per sex, each group receiving diets containing test substance at various doses, continuously for 13 weeks. Ten additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters. At termination, all core-study animals were euthanized by CO2 anesthesia, and complete necropsies were performed. To screen for potential reproductive toxicity, sperm motility and morphology were evaluated at necropsy, and vaginal cytology was evaluated on core-study animals during the week just preceding necropsy. For the 12 d prior to termination, females were subject to vaginal lavage with saline solution. The relative preponderance of leukocytes, nucleated epithelial cells and large squamous epithelial cells were used to identify the stages of the estrual cycle. Sperm motility was evaluated at necropsy and was presented as spermatid heads per total testis and per gram of testis.

Exposure to test substance at dietary concentrations as high as 9.67 mg/L did not affect survival or body weight gains of rats or mice. There were no biologically significant effects noted in hematologic analyses in rats. Mild increases in total bile acids and in serum alkaline phosphatase were noted at various times during the studies in rats receiving the higher dietary concentrations of the test substance. Liver weights were increased in male rats receiving the 10% dietary concentration and in male and female mice receiving diets containing 4.91 or 9.67 mg/L test substance. However, there were no histopathologic lesions associated with these liver changes, nor were there any compound-related morphologic changes in any organ in rats or mice. 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 estrous cycles of rats or mice given diets containing the test substance. Thus, no significant adverse effects of the test substance administration were noted in these studies.

Under the test conditions, a reproductive NOAEC value for male/female rats and mice was determined to be 10% (i.e., ca. 5,000 mg/kg bw/day in rats and 13,000 mg/kg bw/day in mice), after 13 weeks of exposure to castor oil.