Castor oil, ester with glycerol

Endpoint summary

Description of key information

In the absence of repeated dose toxicity data on target substance Castor oil, ester with glycerol an analogue read-across approach was conducted on suitable source substances:

Oral, mouse (OECD 408): NOAEL systemic (male) ≥ 15017 mg/kg bw/day; NOAEL systemic (female) ≥ 16786 mg/kg bw/day

Oral, rat (OECD 408): NOAEL systemic (male) ≥ 5835 mg/kg bw/day; NOAEL systemic (female) ≥ 5725 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

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)

Deviations:

yes

Remarks:

: no ophthalmoscopy, no neurology

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories, Gilroy, CA, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: 22.6 - 23.0 g (males) , 17.2 - 17.7 g (females)
- Housing: individually in Polycarbonate cages lined with heat-treated hardwood chips, covered with polyester filter sheets.
- Diet (Control feed (NIH 07) or diet formulations of castor oil): ad libitum; feeders were changed twice per week throughout the study.
- Water (automatic watering system): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 42 - 72
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
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 amount of feed in a twin-shell blender and blended for 15 minutes to achieve a uniform mix. The homogeneity of castor oil in feed at 10% (100 mg/g) was determined by gravimetric analysis, and blends at 0.5% (5 mg/g) were determined by HPLC analysis. These concentrations of chemical in feed were found to be homogeneously distributed by this mixing procedure. The stability of the 0.5% dose level was determined using HPLC; it was found to be stable for at least 21 days when stored in the dark at 5°C and for 3 days when stored open to air and light in a rodent cage. During the studies, formulated diets were stored for no longer than 3 weeks at 5°C; feed hoppers in the animal cages were changed twice weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 weeks

Frequency of treatment:

daily ad libitum feeding

Dose / conc.:

0.62 other: % (w/w; nominal in diet)

Remarks:

corresponding to 917 and 1153 mg/kg bw/day (actual dose received) in males and females, respectively

Dose / conc.:

1.25 other: % (w/w; nominal in diet)

Remarks:

corresponding to 2022 and 2282 mg/kg bw/day (actual dose received) in males and females, respectively

Dose / conc.:

2.5 other: % (w/w; nominal in diet)

Remarks:

corresponding to 3800 and 5009 mg/kg bw/day (actual dose received) in males and females, respectively

Dose / conc.:

5 other: % (w/w; nominal in diet)

Remarks:

corresponding to 7823 and 9627 mg/kg bw/day (actual dose received) in males and females, respectively

Dose / conc.:

10 other: % (w/w; nominal in diet)

Remarks:

corresponding to 15017and 16786 mg/kg bw/day (actual dose received) in males and females, respectively

No. of animals per sex per dose:

10; 10 additional mice/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters at days 5 and 21.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/ CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: 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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from additional mice at days 5 and 21, and at the end of study
- Anaesthetic used for blood collection: Yes, CO2
- How many animals: 10 additional animals
- Parameters checked: 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: Yes
- Time schedule for collection of blood: from additional mice at days 5 and 21, and at the end of study
- How many animals: 10 additional animals
- Parameters checked: 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).


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Complete histopathology examinations were conducted from the control and 10% dose groups. 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.

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 from the control and 10% dose groups.

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

Description (incidence and severity):

No clinical signs were observed in treated animals compared to controls.

Mortality:

no mortality observed

Description (incidence):

No mortalities were observed in treated animals compared to controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of exposed male mice generally were lower than controls, while mean body weights of exposed females generally were higher. There were no obvious indications that these differences were 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 effects observed

Description (incidence and severity):

No significant differences in average food consumption among each sex were observed, although food consumption by female mice receiving diets containing 10% castor oil was slightly lower than controls.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No changes in parameters of haematology were observed between treated and control animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No changes in parameters of clinical chemistry were observed between treated and control animals.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Liver weights were increased in male and female mice that received diets containing 5% or 10% castor oil. Kidney weights were increased in female mice that received 5% or 10% diets.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Using light microscopy, it was determined that there were no morphologic changes associated with the slight differences between groups in organ weights.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological examination revealed no treatment-related lesions in any organs or tissues of mice exposed to castor oil in the diet.

Histopathological findings: neoplastic:

no effects observed

Details on results:

OTHER FINDINGS
Castor oil exposure produced no adverse effects on any male (testes weight, epididymal sperm motility, density, or testicular spermatid head count) or female (oestrus cycle length, or time spent in each phase of the cycle) reproductive parameter among mice. The low value for sperm motility in control mice was attributed to poor preparative technique.

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % (w/w)

Based on:

test mat.

Remarks:

corresponding to doses of 15017 and 16786 mg/kg bw/day in males and females, respectively, as calculated from the reported food consumption and body weights

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

not specified

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

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)

Deviations:

yes

Remarks:

: no ophthalmoscopy, no neurology

GLP compliance:

yes

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, USA
- Age at study initiation: 6 weeks
- Weight at study initiation: 126 - 132 g (males), 107- 110 g (females)
- Housing: rats: 5 per cage
- Diet (Control feed (NIH 07) or diet formulations of castor oil): ad libitum; feeders were changed twice per week throughout the study.
- Water (automatic watering system): ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 42 - 72
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
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 amount of feed in a twin-shell blender and blended for 15 minutes to achieve a uniform mix. The homogeneity of castor oil in feed at 10% (100 mg/g) was determined by gravimetric analysis, and blends at 0.5% (5 mg/g) were determined by HPLC analysis. These concentrations of chemical in feed were found to be homogeneously distributed by this mixing procedure. The stability of the 0.5% dose level was determined using HPLC; it was found to be stable for at least 21 days when stored in the dark at 5°C and for 3 days when stored open to air and light in a rodent cage. During the studies, formulated diets were stored for no longer than 3 weeks at 5°C; feed hoppers in the animal cages were changed twice weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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 weeks

Frequency of treatment:

daily ad libitum feeding

Dose / conc.:

0.62 other: % (w/w; nominal in diet)

Remarks:

corresponding to 404 and 401 mg/kg bw/day in males and females, respectively (actual dose received)

Dose / conc.:

1.25 other: % (w/w; nominal in diet)

Remarks:

corresponding to 809 and 797 mg/kg bw/day in males and females, respectively (actual dose received)

Dose / conc.:

2.5 other: % (w/w; nominal in diet)

Remarks:

corresponding to 1583 and 1569 mg/kg bw/day in males and females, respectively (actual dose received)

Dose / conc.:

5 other: % (w/w; nominal in diet)

Remarks:

corresponding to 3067 and 3045 mg/kg bw/day in males and females, respectively (actual dose received)

Dose / conc.:

10 other: % (w/w; nominal in diet)

Remarks:

corresponding to 5835 and 5725 mg/kg bw/day in males and females, respectively (actual dose received)

No. of animals per sex per dose:

10; 10 additional rats/sex were included at each dose level for evaluation of hematological and clinical chemistry parameters at days 5 and 21.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS/ CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: 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


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: from additional rats at days 5 and 21, and at the end of study
- Anaesthetic used for blood collection: Yes, CO2
- How many animals: 10 additional animals
- Parameters checked: 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: Yes
- Time schedule for collection of blood: from additional rats at days 5 and 21, and at the end of study
- How many animals: 10 additional animals
- Parameters checked: 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).


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Complete histopathology examinations were conducted on all rats 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.

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 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

Description (incidence and severity):

Group mean body weights of rats receiving diets containing castor oil did not differ significantly from controls. Mean body weights of exposed female rats were slightly lower than the mean body weights of controls but the differences were not dose-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No significant differences in average food consumption among each sex were observed, although food consumption of male and female rats receiving diets containing 10% castor oil was slightly lower than that of controls.

Food efficiency:

not examined

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):

Hematological effects of the castor oil diets among male rats included a slight decrease in MCHC at day 21 in those receiving the 10% diet; a statistically significant decrease in MCV among the 10% group; a decrease in MCH among the 5% and 10% groups; and an increase in platelets among the 1.25%, 5%, and 10% 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% or 10% diets. None of these changes was considered biologically significant.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A treatment- and dose-related increase in the activity of serum alkaline phosphatase was observed in male and female rats at days 5 and 21, and at study termination. Total bile acids were increased among males receiving the higher dietary levels at days 5 and 21 but were not increased at study termination. Other minor changes included increases in albumin observed at study termination in males receiving 5% diets and at day 5 in females receiving 10% diets, and an increase in urea nitrogen at study termination in males that received 0.62% diets and a decrease at day 5 in females that received castor oil at 10% in the diet.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute liver weights and the liver-to-body-weight ratio were increased in male rats that received diets containing 10% castor oil. Heart-to-body-weight ratios were increased in groups of male rats receiving 0.62%, 2.5%, and 10% diets; however, absolute heart weights were not increased, and the differences in body weight ratios were small and not considered treatment related.

In male rats, 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 were no effects on any other male rat reproductive endpoint, or on any female rat reproductive endpoint. Although there was some variation in epididymal weights, 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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Using light microscopy, it was determined there were no morphologic changes associated with the slight differences in organ weights between groups.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

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

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

10 other: % (w/w)

Based on:

test mat.

Remarks:

corresponding to doses of 5835 and 5725 mg/kg bw/day in males and females, respectively, as calculated from the reported food consumption and body weights

Sex:

male/female

Basis for effect level:

other: no overall effects

Key result

Critical effects observed:

no

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 352-426 g (males; mean: 372 g), 192-249 g (females; mean: 224 g)
- Housing: individual in stainless steel cages
- Diet: ad libtum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 - 23.2
- Humidity (%): 48 - 61
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted in appropritate amounts of corn oil for each dose level. Aliquots of the dosing solution corresponding to the amount of daily administration were stored in the dark at 2 - 6 °C. The stability of the dosing solution was 7 days in a refrigerator and 1 day at room temperature. Therefore, the dosing solution was used within 7 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance showed low solubility in water.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): V4N3566

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No details reported.

Duration of treatment / exposure:

males: 42 days (14 days prior to mating and 28 days thereafter)
females: 42-52 days (from 14 days before mating to day 4 of lactation)
satellite males and females: 42 days and 14 days post-exposure observation period

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 females in control and test groups
7 males in control and 1000 mg/kg bw groups
12 males in 100 and 300 mg/kg bw groups
5 animals per sex in satellite control and 1000 mg/kg bw/day groups (in addition to number listed above)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Two dose range finding studies were performed. 2000 mg/kg bw of test substance was administered for 3 days in male and female rats. No abnormalities were found in general condition and body weight. The second study was performed at dose levels of 0, 330, 100, 300 and 1000 mg/kg bw/day for 14 days. No abnormalities of general condition, body weight, food consumption, hematological findings, blood biochemical findings, gross pathology and organ weight were found. Therefore, 1000 mg/kg bw/day was selected as the highest dose.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Day 1 (before administration), 7, 14, 21, 28, 35 and 42 (before sacrifice)
Females: Day 1 (before administration), 7, 14, during pregnancy on Day 0, 7 14 and 21, during lactation on Days 0 and 4 (before sacrifice)
Satellite males and females: Day 1 (before administration), 7, 14, 21, 28, 35, 42, 49 (Day 7 of recovery period) and 56 (Day 14 of recovery period, before sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after last application
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, Platelets, Reticulocytes, PT, APTT, WBC, Differential leukocytes: Lymphocytes, Neutrophils, Eosinophils, Basophils, Monocytes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after last application
- Animals fasted: Yes
- How many animals: 5 per group
- Parameters checked: AST, ALT, ALP, γ-GTP, T-protein, Albumin, A/G, T-bilirubin, BUN, Creatinine, Glucose, T-cholesterol, Triglyceride, Na, K, Cl, Ca, P, LDH, choline esterase

URINALYSIS: Yes (males)
- Time schedule for collection of urine: Day 37 during administration period or Day 9 during recovery period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Colour, cloudiness, water consumed, volume, specific gravity, Na, K, pH, Protein, Glucose, Ketone body, Bilirubin, Occult blood, Urobilinogen, Epithelial cells, Erythrocytes, Leukocytes, Casts, Crystals, Fat

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
Males: on Week 6 of the administration period
Females: on Week 6 of the administration period
Males and females of satellite groups: on Week 6 of the administration period and on Week 2 of the recovery period
- Dose groups that were examined: all
- Battery of functions tested: hearing reaction, eye sight reaction, sense of touch reaction, pain reaction, pupil reflex, pinna reflex, ipsilateral flexor reaction, eyelid reflex, righting reflex

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Body surface, mucous membranes and internal organs

HISTOPATHOLOGY: Yes
Brain, pituitary, thyroid, thymus, lung trachea (after liquid immersion fixation), stomach, intestines, heart, liver, spleen, kidney, adrenal gland, bladder, testis, epididymis, prostate, seminal vesicles, ovaries, uterus, spinal cord (cervical, thoracic, lumbar), sciatic nerve, bone marrow (femur), lymph nodes (cervical lymph node, mesenteric lymph nodes), mammary gland, and other gross abnormalities
Spermatogenic cycle (Stage II, III, V, VII, and XII) was also investigated.

SACRIFICE
- Males: on Day 43
- Females: on Day 5 of lactation
- Females (unsuccessful mating): on Day 53
- Females (mated but non-pregnant): on Day 5 after scheduled delivery
- Satellite males and females: on Day 15 of the recovery period

Other examinations:

Organ weight: brain, liver, kidney, spleen, heart, thymus, thyroid, pituitary, adrenal, testis, sminal vesicle, epididymis
Estrous cycle
Number of ovarian corpora lutea
Number of uterine implantation
Observation of pups

Statistics:

ANOVA, Barlettm Kruskal-Willis, Dunnett, F test, Studen t-test, Aspin-Welch t-test, Mann-Whitney U-test, Fisher's exact test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, males): significant increase was observed (non adverse)

No change of body weight and weight gain was observed during the administration period. During the recovery period, a significant increase in body weight was noted in males at 1000 mg/kg bw/day. This was caused by a tendency of the control group animals to lose weight. One male in control group showed a significant decrease in body weight during the recovery period. However, no other abnormalities were observed in this male.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, females): significant increase on Day 14 of administration (non adverse)

No change was observed during the administration period in the test groups. A significant increase in food consumption was found in satellite females of the 1000 mg/kg bw/day group on Day 14 of administration. However, it was regarded as an incidental finding since the food consumption of the corresponding control group was relatively small on that day. Therefore, this change was not regarded as a compound-related effect.

Food efficiency:

not examined

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):

100 and 300 mg/kg bw/day (males): decrease in APTT without dose-dependency after administration period (non adverse)

No changes were observed. After administration, a significant decrease in APTT was observed in males of the 100 and 300 mg/kg bw/day groups in comparison with control group. However, this change was not regarded as compound-related, because no dose-dependency was observed and this was within reference range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day (females): significant increase in organic P in females after administration period without dose-dependency (non adverse); 1000 mg/kg bw/day (satellite group, females): significant increase in choline esterase (non adverse)

A significant decrease in inorganic P was observed in females of the 300 and 1000 mg/kg bw/day groups after administration. However, this change was not regarded as compound-related, since a dose-dependency was not observed and almost all the individual data were within the reference ranges except for one female of 300 mg/kg bw/day.
Choline esterase was increased in females of 1000 mg/kg bw/day after the recovery period but this was within the reference range. This change was not compound-related effect since this change was slightly and no change was observed after the administration period.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No significant changes were found during the administration and recovery periods.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No abnormality was observed for the sensory/reflex function, landing foot, grip strength and motor activity during the administration period. During the recovery period, motor activity increased from 0 to 60 min but not from 0 to 30 min in females dosed 1000 mg/kg bw/day. This effect was not compound-related since the observed change was slight and no change was observed during and after the administration period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

100 mg/kg bw/day: significant decrease in absolute weight of seminal vesicles in male and significant decrease in relative weight of spleen in females after administration period (non adverse)

After the administration period, a significant decrease in the absolute weight of seminal vesicles in males given and a significant decrease in relative weight of spleen in females were observed in the 100 mg/kg bw/day group. However, this change was not compound-related because no abnormalities were found at histopathological examination and there was no dose-dependency.
After the recovery period, a significant decrease in relative weight of pituitary in males of the 1000 mg/kg bw/day group and relative weight of thyroid in females of the 1000 mg/kg group bw/day was observed. Nevertheless, this effect was not compound-related because no abnormality was reported regarding these organs after administration period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: mass in subcutis in one female after administration period (not treatment-related)

No test substance-related changes were found. No abnormalities were found in breeding pairs which were not successful at mating and in those which were successful at mating but not pregnant.
After administration period, reddish thymus was noted in one male of the control group and subcutis mass was found in one female of 300 mg/kg by/day group (see also HISTOPATHOLOGY: NEOPLASTIC). After the recovery period, larger spleen and capsular thickening in spleen was found in one male and reddish area in thymus was observed in one female at 1000 mg/kg bw/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

control and 1000 mg/kg bw/day: after administration period, low incidence of commonly found microscopic changes, evenly distributed between groups (not treatment-related)

No test substance-related changes were observed. There were also no changes regarding spermatogenic cycle.
Myocardial degeneration/fibrosis, foam cell accumulation in lung, mineralization in artery in lung, fatty degeneration of hepatocyte, microgranuloma in liver, solitary cyst in kidney, hyaline cast in kidney, lymphocyte infiltration in cortex of kidney, mineralization of cortico-medullary junction in kidney, fibrosis of cortex in kidney and haemorrhage in thymus were observed both in the control and 1000 mg/kg bw/day groups or only in the control group with low incidence. Hyaline droplet of proximal tubular epithelium in kidney was found in all males of the control and 1000 mg/kg bw/day groups. Brown deposit pigment and extramedullary haematopoiesis in spleen were found in all males and females of the control and 1000 mg/kg bw/day groups. However, there were no differences between control and test group. In the 1000 mg/kg bw/group, lymphocyte interstitium infiltration in prostate was found in one male, interstitial focal inflammation in lung and focal necrosis in liver was observed in one female. These changes occur naturally and were not compound-related.
No abnormalities were found in uterus and ovary in the non-pregnant females and the unsuccessful copulation females of the control and 1000 mg/kg bw/day groups, respectively. Degeneration of seminiferous tubules of testis, decrease in sperm and atrophy in prostate was observed in one control male.

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

300 mg/kg bw/day: after administration period, benign fibroadenoma of the mammary gland in one female (not treatment-related)

Mass on abdomen was found in a female after 40 days of administration in the 300 mg/kg bw/day group. However, this subcutaneous tumour of the mammary gland was a benign fibroadenoma and generated naturally.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Key result

Critical effects observed:

no

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:

The available information comprises an adequate and reliable study (Klimisch score 1) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to endpoint discussion for further details). The selected to study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

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

Justification for read-across

There are no studies on the repeated dose toxicity of Castor oil, ester with glycerol available. The assessment of repeated dose toxicity was based on studies conducted with two source substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Repeated dose toxicity, oral, subchronic

CAS 8001-79-4 (source substance)

The subchronic oral toxicity of Castor oil was investigated in male and female Fischer 344 rats in a GLP-conform study similar to OECD guideline 408 (Key, 1992). The test substance was administered daily ad libitum for a period of 13 weeks to groups of 20 animals per sex at dietary concentrations of 0.62, 1.25, 2.50, 5 and 10% (w/w), corresponding to reported doses of 404, 809, 1583, 3067 and 5835 mg/kg bw/day in males and 401, 797, 1569, 3045, 5725 mg/kg bw/day in females, respectively. A similar constituted control group received the plain diet. Ten of the 20 animals per sex and group were used to analyse haematological and clinical chemistry parameters on Days 5 and 21 and at the end of the study. During the study period, no mortalities and no clinical signs were observed. Body weights and food consumption in treated animals were comparable to controls. No biologically relevant changes were observed in parameters of clinical chemistry and haematology on Days 5 and 21, and at the end of the study. The absolute and relative liver weights were increased at a dose level of 5835 mg/kg bw/day. An increase in relative heart weight in males was noted at 404, 1583 and 5835 mg/kg bw/day. Furthermore, a slight decrease in epididymal weight was observed in male animals of the middle and high dose groups. However, these effects were not considered to be treatment-related, since they occurred in the absence of dose-dependency and any correlating histopathological changes. Macroscopic and microscopic examinations did not reveal any substance-related effects in animals of all dose groups. Based on the overall effects of the study, a NOAEL of ≥ 5835 and 5725 mg/kg bw/day for male and female Fischer 344 rats, respectively, was derived.

In a further subchronic oral toxicity study performed similar to OECD guideline 408, the test substance was administered to B6C3F1 mice at dietary concentrations of 0.62, 1.25, 2.50, 5 and 10% (w/w), corresponding to reported doses of 917, 2022, 3800, 7823, 15017 mg/kg bw/day in males and 1153, 2282, 5009, 9627, 16786 mg/kg bw/day in females, respectively (Supporting, 1992).20 animals per sex and group received the test substance in the diet or plain diet (controls) daily ad libitum for 13 weeks. Ten of the 20 animals per sex and group were used to analyse haematological and clinical chemistry parameters on Days 5 and 21. At study termination, no mortalities, no clinical signs and no biologically relevant changes in body weights and food consumption were observed in all treated animals compared to controls. No effects on parameters of clinical chemistry and haematology were observed in treated all animals compared to controls. At 9627 and 16786 mg/kg bw/day, liver weights were increased in both sexes, whereas at the same dose levels kidney weights were only increased in females. Since the effects were marginal and not supported by histopathological findings, they were considered as non-adverse. Gross pathology and histopathology did not reveal any substance-related findings in animals of all dose groups. Based on the results of this study, the NOAEL for male and female B6C3F1 mice was set at ≥ 15017 and 16786 mg/kg bw/day, respectively.

Repeated dose toxicity, oral, subacute

CAS 111-03-5 (source substance)

A GLP-compliant subacute oral toxicity study according to OECD 422 was performed with 2,3-dihydroxypropyl oleate at doses of 100, 300 and 1000 mg/kg bw/day (Supporting, 2005). Male and female Sprague Dawley rats (12 per sex and group, except for 1000 mg/kg bw/day: only 7 males) received the test substance in corn oil once daily via gavage. A control group, consisting of 7 males and 12 females, was treated with the vehicle alone. The duration of treatment was 42 days (14 days prior to mating and 28 days thereafter) in males and 42-52 days (from 14 days before mating to day 4 of lactation) in females, respectively. Satellite groups of 5 animals per sex, each for the control and test groups, were used to investigate reversibility of effects during a 14-day post-exposure recovery period. No mortality and no clinical signs and effects on neurobehaviour were observed during the whole study period. No adverse effects on body weight development were observed during treatment and recovery period. The analysis of clinical, haematological and urinary parameters did not reveal any dose-dependent and toxicologically relevant changes in treated animals compared to controls. At 100 mg/kg bw/day, a significant decrease in absolute weight of seminal vesicles in males and a significant decrease in relative weight of spleen in females were observed at necropsy. Since these effects did not follow a dose-dependent relationship and were not accompanied by any histopathological changes in the respective organs, they were not considered to be toxicologically relevant. A decrease in relative weights of pituitary in males and thyroid in females was observed at 1000 mg/kg bw/day at the end of the recovery period. Since no abnormities were reported for these organs after the administration period, they were considered as not compound-related. No test substance-related changes were found at gross pathology. A low incidence of commonly found microscopic changes was observed, which was evenly distributed between all groups and therefore considered as incidental findings. Although one female showed a subcutaneous tumor of the mammary gland after 40 days exposure to 300 mg/kg bw/day, this benign fibroadenoma was considered to be generated naturally, since no effects were observed at the higher dose levels. Based on the overall effects observed in this subacute study, a NOAEL of 1000 mg/kg bw/day for male and female Sprague Dawley rats was derived.

Overall conclusion for repeated dose toxicity

The available data on the repeated dose toxicity of source substances comprise a sub-acute study in rats and a sub-chronic study in rats and mice via the oral route. In all available studies NOAEL values for oral repeated dose toxicity were at or well above the currently applied limit dose value of 1000 mg/kg bw/day. Thus, no hazard after repeated oral exposure was identified.

Therefore, as the available data did not identify any hazard for oral repeated dose toxicity, Castor oil, ester with glycerol is not expected to be toxic after repeated oral exposure.

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that "substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Castor oil, ester with glycerol, data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach , the available data on repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.