Hydroxyoctadecanoic acid, monoester with glycerol

Endpoint summary

Description of key information

No adverse effects observed in 13-week dietary study of castor oil, a structural analogue of the registered substance.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on species / strain selection:

F344/N

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Simonsen Laboratories, Gilroy, CA
Age: 6 weeks
Quarantine: held 14-15 days
Rats housed 5/cage, mice housed individually
Cage type: Polycarbonate with heat-treated wood chips
Diet: NIH-07 diet, ad libitum. Water ad libitum
Temp--68-76°F; relative humidity--42-72%; fluorescent light 12 h/d; 10 room air changes/h.
Observations: Animals were observed twice daily, weighed initially and weekly thereafter.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared every 21 days. Feed within animal cages was changed every 3 days.
- Mixing appropriate amounts with (Type of food): NIH07, ad lib
- Storage temperature of food: at 5 degrees C, in the dark

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: No. By cage, with 5 animals/cage.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Purity analysis indicated that it was consistent with the USP specifications and the reported composition for castor oil: Analysis was conducted by Midwest Research Institute (MRI), in Kansas City, MO, utilizing infrared, UV/Vis and NMR spectroscopy, Karl Fischer water analysis, TLC and HPLC, and a battery of USP standard analyses for castor oil.
The stability of the test material during the toxicology studies was monitored by determination of peroxide content and by HPLC. The substance was stable during the course of the studies.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

equivalent to 0.62% in the diet. Actual: male/female: 404/401 mg/kg bw/d

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

equivalent to 1.25% in the diet. Actual: male/female: 809/797 mg/kg bw/d

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

equivalent to 2.5% in the diet. Actual: male/female: 1583/1569 mg/kg bw/d

Dose / conc.:

3 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 5.0% in the diet. Actual: male/female: 3067/3045 mg/kg bw/d

Dose / conc.:

6 000 mg/kg bw/day (nominal)

Remarks:

equivalent to 10% in the diet. Acutal: male/:females: 5835/5725 mg/kg bw/d

No. of animals per sex per dose:

10 rats/sex/group in the core study, plus 10 rats/dose/sex were included for hematological and clinical chemistry parameters at days 5 and 21.

Control animals:

yes, plain diet

Details on study design:

The core study was conducted with groups of 10 rats and 10 mice per sex, each group receiving diets containing 0, 0.62%, 1.25%, 2.5%, 5.0% or 10% castor oil, continuously for 13 weeks. Ten additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters. At days 5 and 21, these animals were anesthetized with CO2, and blood was collected from the orbital sinus. These animals were killed following the blood collection on day 21. Blood samples for hematology and clinical chemistry also were collected from core-study rats at study termination.


Clinical Examinations, Supplemental Studies and Pathology

At the study termination, all core-study animals were euthanized by CO2 anesthesia, and complete necropsies were performed. Complete histopathology examinations were conducted on all rats and mice from the control and 10% dose groups. Livers were examined from male rats in all other dose groups; histologic sections of gross lesions were examined from all rats. 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.

On days 5 and 21, and at sacrifice, blood samples were collected from the orbital sinus of non-fasted animals under CO2 anesthesia. Hematology parameters were determined on a Baker 9000 automated hematology analyzer (J.T. Baker, Phillipsburg, NJ) and included:
Red blood cell (RBC) count, red blood cell morphologic assessment, hematocrit (HCT), hemoglobin concentration (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cell count (WBC), white blood cell differential count, reticulocyte count (absolute), and platelet counts (absolute). Clinical chemistry assays, performed with a Centrifichem 600 (J.T. Baker, Phillipsburg, NJ) using commercially available kits and standard methods developed for this instrument, included alkaline phosphatase activity (ALP), albumin (ALB), urea nitrogen (UN), creatinine (CREA), alanine aminotransferase activity (ALT), total bile acids (TBA), sorbitol dehydrogenase activity (SDH), total protein (TP), and creatinine kinase (CK).

Upon completion of the histologic evaluation by the laboratory pathologist, the slides, paraffin blocks, and residual wet tissues were sent to the NTP Archives for inventory, slide/block match, and wet tissue audit. The slides, individual animal data records, and pathology tables were sent to an independent pathology laboratory where quality assessment was performed; the results were reviewed and evaluated by the NTP.

Reproductive Toxicity Screen

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, following published procedures (Morrissey et al., 1988). For the 12 days prior to termination, females were subject to vaginal lavage with saline. The aspirated cells were air-dried onto slides, stained with Toluidine Blue O, and coverslipped. 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 as follows: The left epididymis was removed and quickly weighed; the cauda epididymis was removed at the junction of the vas deferens and the corpus epididymis, then weighed. A small cut was made in the distal cauda epididymis. The sperm that were removed from the epididymis were dispersed and the number of moving and non-moving sperm were counted in 5 fields of 30 sperm or less on each slide. After sperm sampling for motility evaluation, the cauda was placed in phosphate buffered saline (PBS), and gently chopped with a razor blade. The solution was mixed gently and heat-fixed at 65°C. Sperm density was then determined using a hemocytometer.

The left testis was frozen and stored. After thawing, testicular spermatid head count was determined by removing the tunica albuginea and homogenizing the testis in PBS containing 10% DMSO. Homogenization spermatid nuclei were enumerated using a hemocytometer; the data were expressed as spermatid heads per total testis and per gram of testis.

Positive control:

None

Observations and examinations performed and frequency:

Animals were observed twice daily.

Sacrifice and pathology:

CO2 asphyxiation was followed by retroorbital blood sampling and complete necropsies for the control and high doses.

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, forestomach 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 10% 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).

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

Description (incidence and severity):

No deviations from expected food consumption/compound intake was observed.

Food efficiency:

not specified

Description (incidence and severity):

No description of caloric breakdown by dose.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In males, a statistically significant decrease in MCV in the 10% group was found, but was not biologically significant. Minor effects: in males of the 10% group at day 90, a slight decrease in MCH along with a transient decrease in MCHC in the 10% group males at day 21. At 90 days, the platelet count in males of the 1.25, 5 and 10% groups was significantly increased. These variations were evaluated as not biologically significant by the study authors.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males and females, a dose-related increase in serum alkaline phosphatase activity was found at days 5, 21 and 90. At days 5, 21 and 90, in males and females, there was a consistent dose-related increase in serum alkaline phosphatase activity in the 5 and 10% groups. Total bile acids were also statistically significantly elevated in males of the 5 and 10% groups at 5, 21 and 90 days. This is likely an adaptation to increased absorption and metabolism of lipids from the intestinal tract.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute liver weight and liver-to-body weight ratio were statistically significantly increased in male rats receiving 10% castor oil. Relative heart ratios were increased in some doses but not in a dose-related manner; as absolute heart weights were not increased, they were not considered treatment related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No gross pathologic lesions noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histopathologic lesions in liver or any other organ.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Reproductive function endpoints; slight decrease in epididymal weight (<10% in magnitude). This is not considered evidence of reproductive toxicity.

Details on results:

No toxicologically relevant adverse effects were observed in any treatment group.

In males in the highest dose group (10%), there was a statistically significant decrease in MCV ; this was not considered biologically significant. Also in this group, there was a slight decrease in MCH and the MCHC was transiently decreased at day 21. Other minor effects were observed in hematologic parameters: at 90 days, the platelet count in males of the 1.25, 5 and 10% groups was significantly increased. These variations were evaluated as not biologically significant by the study authors.

Serum alkaline phosphatase activity was increased in a treatment- and dose-related manner at days 5, 21 and 90, in both males and females in the 5 and 10% groups. Total bile acids were also statistically significantly elevated in males of the 5 and 10% groups at 5, 21 and 90 days. This is likely an adaptation to increased absorption and metabolism of lipids from the intestinal tract.

Absolute liver weight and liver-to-body ratio were increased in male rats at the high dose (10%). In males, there was a slight decrease in epididymal weight (< 10%) in the middle and high dose groups, but this was not dose-related. It is concluded that there was little or no evidence of any reproductive toxicity associated with castor oil exposure.

Histological examination revealed an absence of compound related lesions in any organ or tissue of rat exposed to castor oil in the diet.

The NOAEL was determined by the authors to be 10% in the diet, equivalent in females to 5725 mg/kg bw/d, and in males to 5835 mg/kg bw/d.

Key result

Dose descriptor:

NOAEL

Effect level:

5 725 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No adverse effects at the highest dose (10% in the diet, 5725 mg/kg bw/d in females (actual), 5835 mg/kg bw/d in males (actual)

Key result

Critical effects observed:

no

There were no observed loose or wet feces associated with castor oil intake in rats, likely due to its presence in food ingested over a relatively prolonged time period.  Survival, food consumption, and body weights in treated animals were comparable to those of controls. Changes in serum liver enzyme levels reflect metabolic adaptation to the high lipid diet ingested. There were no gross or histopathologic lesions associated with the liver changes, nor were there any compound-related morphologic changes in any organ examined. The administration of diets containing up to 10% castor oil was not associated with toxicity to any specific organ, organ system or tissue, under the conditions of this study.

TABLE 3

Survival and Average Food Feed Studies of Castor Oil

and

Compound

Consumption

of F344/N

Rats

in

the

13-Week

Dose

Mean

BodyWeight

(grams)

Final Weight Relative to Controls (%)

FeedConsumption

Compound Consumption

(% in feed)

Survival

(a)

Initial

Final

Change

(b)

(c)

MALE 0 0.62 1.25 2.5 5.0 10.0   FEMALE 0 0.62 1.25 2.5 5.0 10.0

10/10 10/10 10/10 10/10 10/10 10/10   10/10 10/10 10/10 10/10 10/10 10/10

132 130 126 131 131 129   108 108 107 109 110 108

364 346 359 356 351 353   208 202 205 202 206 197

233 216 233 226 220 224   100 95 97 93 96 89

95.0 98.6 97.8 96.4 97.0     97.1 98.6 97.1 99.0 94.7

65 65 65 63 61 58   64 65 64 63 61 57

0 404 809 1583 3067 5835   0 401 797 1569 3045 5725

(a) (b) (c)

Number surviving/number initially in group. Averagegrams food consumed per kg body weight per day. Average mg compound consumed per kg body weight per day.

Conclusions:

The administration of diets containing up to 10% (w/w) of castor oil to F344 rats for 13 weeks was not associated with toxicity to any specific organ, organ system or tissue under the conditions of this study. The NOAEL is greater than 10%, equivalent to 5725 mg/kg bw/d in females and 5835 mg/kg bw/d in males (actual). The read-across approach from castor oil to GMHS is acceptable for the purposes of classification and labelling, and filling registration data requirements.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

5 725 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

adequate

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity testing has been undertaken on structural analogues, the triglycerides castor oil and hydrogenated castor oil. No adverse effects were observed in rats given dietary castor oil for 13 weeks. The NOAEL was > 5000 mg/kg bw/d, specifically 5725 mg/kg bw/d in females and 5835 mg/kg bw/d in males. In an older series of 13- and 16-week dietary studies by Binder, et al., 1970, hydrogenated castor oil showed no adverse effects in Slonaker rats after ingesting 10% in the diet other than reduced growth rate (attributed to lower caloric density and lower bioavailability). Due to this confounding issue, the NOAEL was 1% in the diet. No data are available on mg/kg/day equivalencies. Perkins, et al., 1961, found no adverse effects after 60 days of feeding hydrogenated castor oil to rats. The expert consulting group, DHI, on behalf of ECHA, determinined that the existing body of knowledge on castor oil was adequate for risk assessment, and no further testing was needed.

The most reliable and precise NOAEL, from the 1992 NTP study, provides a NOAEL of 5725 mg/kg bw/d in female rats.

Justification for classification or non-classification

No systemic toxicity was seen with castor oil or hydrogenated castor oil in repeated dose toxicity studies of at least 13-weeks duration, suggesting a NOAEL of 5725 mg/kg bw/d or higher. Review of the body of existing data on the repeated dose toxicity of castor oil by the expert consulting group, DHI, on behalf of ECHA, resulted in a decision to confirm castor oil on Annex IV (later moved to Annex V).  This reflects the decision that the NOAEL is sufficiently high to warrant no concern for toxicologic effects from repeated dose exposure.