Registration Dossier

Administrative data

Description of key information

Oral (OECD 408), rat: NOAEL (systemic) ≥  1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study, tested with the source substance castor Oil (CAS 8001-79-4). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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 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
Remarks:
Doses / Concentrations:
0.62, 1.25, 2.50, 5.00, 10.0 % (w/w)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
404, 809, 1583, 3067 and 5835 mg/kg bw/day
Basis:
other: actual ingested: male rats
Remarks:
Doses / Concentrations:
401, 797, 1569, 3045, 5725 mg/kg bw/day
Basis:
other: actual ingested: female rats
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
Food consumption and compound intake (if feeding study):
no effects observed
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):
no adverse biologically significant effects
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
no adverse biologically significant effects
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):
no adverse biologically significant effects
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
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 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.

HAEMATOLOGY
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 CHEMISTRY
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.

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

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. Histopathologic examination revealed an absence of compound-related lesions in any organ or tissue of rats exposed to castor oil in the diet.
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:
not specified
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Feb - 07 April 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). According to the ECHA guidance document ‘Practical guide 6: How to report read-across and categories' (ECHA, 2012), the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to
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:
other: Crl:WI (Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277 - 353 g (males) and 183 - 225 g (females)
- Housing: 5 animals of the same sex per cage in Macrolon cages (MIV type). Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, UK) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 h prior to dosing and were homogenised to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

DOSE VOLUME:
5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 h.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 5 females (main study)
5 males and 5 females (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 10-day dose range finding study.
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were conducted during the treatment phase only. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period (except for males). Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: White blood cells, Differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: ALAT, ASAT, ALP, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the first Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual animals, using a computerized monitoring system; Pearson Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually. The assigned animals were tested during week 4 of treatment (all before blood sampling). Since no treatment-related findings were noted, the functional observation tests and motor activity measurements were not extended to all animals at the end of the recovery phase.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- From the first 5 Main males (randomly selected at allocation), the 5 Main females and all
Recovery animals per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Ovaries, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid.

- From all remaining males: Epididymides, Testes

Since no toxicologically relevant effect was noted on organ weights of Main females, no organ weights were collected from Repro females.
Sacrifice and pathology:
DAY OF NERCROPSY:
- Main animals: Following completion of a minimum of 28 days of dose administration.
- Recovery animals: Following completion of a minimum of 28 days of dose administration and a recovery period of 14 days.
One animal was euthanised in extremis.

GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
From the first 5 Main animals/sex/group, all Recovery animals, the selected Repro females/group (with live offspring) and animal no.117 that was killed in extremis (Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination):
Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (including optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes1, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

From all remaining animals and females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.











Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.
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: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
No clinical signs of toxicity were noted that were attributable to treatment.

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Female no. 117 (1000 mg/kg bw/day) was killed in extremis on Day 17 post-coitum. Microscopic examination revealed a marked granuloma in the bronchus region containing macrophages surrounding food particles and with central areas of necrosis. These findings were indicative of a gavage accident.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day.
Any statistically significant changes in body weight gain observed among males and females during the treatment or recovery period were considered to be of no toxicological relevance since the changes occurred in the absence of a dose-related trend and/or were of a slight and/or temporary nature. These changes consisted of a higher body weight gain for males at 1000 mg/kg bw/day during the recovery phase, a lower body weight gain of Repro females at 1000 mg/kg bw/day on Day 11 of the post coitum period, and a higher body weight gain of Main females at 100 mg/kg bw/day over treatment Days 8-22.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time in males at 1000 mg/kg bw/day and lower relative lymphocyte counts in females at 300 mg/kg bw/day at the end of treatment were considered to be of no toxicological relevance. These changes were absent at the end of the recovery period, occurred in the absence of a (clear) treatment-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. At the end of treatment these changes consisted of lower alanine aminotransferase in males at 300 and 1000 mg/kg bw/day, and higher chloride levels in males at 100 mg/kg bw/day. Changes at 1000 mg/kg bw/day at the end of the recovery phase included lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in males.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg bw/day had significantly lower high sensor counts. Since the range of high sensor values encountered at 300 mg/kg bw/day was similar to that observed in the control group and no dose-related trend was noted, no toxicological relevance was ascribed to this variation. A notable variation in high sensor counts was recorded for females at 1000 mg/kg bw/day. Since the range of values at this dose was essentially similar to that observed in the control group, it was considered that no toxicologically significant effect on high sensor counts had occurred in females at 1000 mg/kg bw/day.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. Also, no histopathological correlates were noted to support these changes. These changes consisted of a lower spleen to body weight ratio in males at 1000 mg/kg bw/day at the end of the recovery phase, a higher spleen weight and spleen to body weight ratio in females at 100 mg/kg bw/day and the end of treatment, lower heart weight in females at 1000 mg/kg bw/day, higher heart to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment, lower adrenal weight and/or adrenal to body weight ratio in females at 300 and 1000 mg/kg bw/day at the end of treatment, and a lower ovary and ovary to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg bw/day euthanized in extremis (no. 117) showed a hard, greenish nodule on the right medial lobe of the lung, dark red discolouration of the left ovary, enlarged adrenal glands, reduced size of the thymus, enlargement and greenish discolouration of the bronchial lymph node and pleura grown together with the lungs. A watery-clear cyst was found for a single control Repro female (no. 89). This finding was corroborated with a cyst found in the cervix found upon histopathological examination, which likely contributed to this animal’s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes, epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and their incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100 mg/kg bw/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a subsequent extensive epididymal oligospermia, which accounted for its infertility. This was considered to be a spontaneous abnormality with no likely relationship to the test item. No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile animals which could account for their infertility. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
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: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.
Key result
Critical effects observed:
not specified
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 Nov 1984 - 21 Jan 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study with acceptable restrictions. Lack of test material details, no analysis of urine and neurobehaviour.
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
lack of test material details, no analysis of urine and neurobehaviour
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
lack of test material details, no analysis of urine and neurobehaviour
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River WIGA, Sulzfeld, Germany.
- Age at study initiation: 4 weeks
- Weight at study initiation: 52-69 g (males) and 54-67 g (females)
- Housing: animals were housed in groups of 2-3 animals in Makrolon type III cages on soft wood bedding
- Diet: Altromin 1324 DK (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 59-67.5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Nov 1984 To: 19 Dec 1984
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared freshly on the day of application. The test substance was grind with a mortar before adding peanut oil.

VEHICLE
- Purity: DAB 7
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 5 days/week
Remarks:
Doses / Concentrations:
100, 500 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 (main study)
5 (satellite low and mid-dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Yielding of a high security range.
- Post-exposure recovery period in satellite groups: 33 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on Day 28
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 28
- How many animals: all animals
- Parameters checked: erythrocytes, haematocrit, haemoglobin concentration, mean cell volume, leukocyte count, thrombocyte count and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 28
- How many animals: all animals
- Parameters checked: urea, creatinine, sodium, potassium, glucose, calcium, alkaline phosphatase, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, gamma-glutamyl transferase, cholesterol, total protein, chloride, bilirubin

ORGAN WEIGHTS:
- Thyroid gland, adrenal gland, thymus, spleen, heart, kidneys, brain, testes, liver
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals. Liver, thyroid gland, kidneys, adrenals, testes, uterus, ovaries, thymus, spleen, brain and heart were examined.
HISTOPATHOLOGY: Yes, from animals of vehicle control and highest dose group: Aorta thoracica, eyes, small and large intestine, glandular stomach, brain, urinary bladder, skin, heart, testes, liver, trachea, lung, maxillary lymph nodes, mesenterial lymph nodes, spleen, epididymis, adrenal gland, peripheral nerv, kidneys, ovary, pancreas, prostate, vesicular gland, thyroid gland, salivary gland, oesophagus, sceletal muscle, thymus, uterus, stomach, tongue. Target organs of digestive tract were histopathologically analysed from all animal groups.
Statistics:
t-test, U-test
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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
high-dose: reduction of HT-value (m), reduction of stab leucocytes (f); mid-dose: reduction of HT-value and reduction of leucocyte count (m), low-dose: increase of thrombocyte and leucocyte count (m), increase leucocyte count (f); non-adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 1 under "Any other information on results incl. tables".
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see Table 2 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Table 3 under "Any other information on results incl. tables".
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived the foreseen number of applications without substance-related symptoms. There were a few individual observations, which appeared at different phases of the study and were considered to be not treatment-related: one male in the control group and one female in the high-dose group showed alopecia, one female in the mid-dose group showed chromodacryorrhoea.
One male in the high-dose group died during narcosis for blood sampling at the end of the exposure period.

BODY WEIGHT AND WEIGHT GAIN
No effects were observed in group mean body weight (gain) over the whole study period.

FOOD CONSUMPTION
During the first week, a slight but statistically significant decrease in mean group food intake was observed in females of the low-dose group compared to the control group (17 vs. 18 g/rat/day), which was considered to be not toxicologically relevant. No further effects were observed in mean group food intake.

WATER CONSUMPTION
No effects were observed in group mean water intake over the whole study period.

OPHTHALMOSCOPIC EXAMINATION
No effects were observed.

HAEMATOLOGY
Low-dose males showed a slight increase in thrombocytes and lymphocytes; low-dose females showed a slight increase in leukocytes. A slight decrease in haematocrit and leukocytes and a slight increase in lymphocytes were observed in males of the mid-dose group; no effects were observed in mid-dose females. In the high-dose group, haematocrit and rod-shaped leukocytes were slightly decreased in males, and leukocytes were slightly increased in females.
Overall, haematological parameters, group and individual values did not allow the identification of a substance-related toxic effect.

CLINICAL CHEMISTRY
In low-dose males, GOT and GPT values were decreased and bilirubin values were increased; no effects on females were observed. Males in the mid-dose group showed a slight decrease in GOT and GPT as well as a decrease in calcium, while females presented a slight increase in protein and cholesterol and slight decrease in chloride. In males of the high-dose group, there was a decrease in GOT, an increase in sodium without concomitant increase in chloride, and a slight decrease in bilirubin. High-dose females showed a slight decrease in GOT and a decrease in chloride.
Overall, none of the above mentioned changes in biochemical parameters could be attributed to the test substance, since there was no dose-dependency and individual values were (with a few not dose-dependent exceptions) within the normal ranges for this species and age.

ORGAN WEIGHTS
Absolute organ weights:
Males in the low-dose group showed a slight decrease in adrenals weight. No effects were observed in low-dose females. In the mid-dose group, males showed a slight decrease in thymus weight and females showed a slight increase in brain weight. A slight decrease in thymus weight was also observed in high-dose males. High-dose females showed no effects in absolute organ weights.
Relative organ weights:
There was only a slight decrease in thymus weight in the mid-dose males.
Overall, no substance- and dose-dependent effects were observed.

GROSS PATHOLOGY
There were no substance- and dose-dependent findings. Sporadic findings included hydrometra, hydronephrosis, kidney cysts, slight redness of the stomach mucosa and inflammation of the forestomach mucosa.

HISTOPATHOLOGY: NON-NEOPLASTIC
Sporadic findings across control and treatment groups included slight interstitial inflammation in the lung, prostate inflammation, slight changes in the forestomach mucosa and increased haematopoiesis in the liver parenchyma.
In the mid- and high-dose groups, non-inflammatory dilatation of lymph vessels in the small intestine as well as foreign body giant cells and vacuolization were found in Peyer Plaques. This observed dilation of lymph vessels and foreign body giant cells (fused macrophages) in the small intestine can be interpreted as adaptive responses to a high dose of triglycerides, as the upper intestinal tract is responsible for triglyceride resorption. The histopathological findings indicated an incomplete enteral resorption. Other parts of the gastrointestinal tracts including the mesenteric lymph nodes were without treatment-related findings.
No other treatment related abnormalities were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Critical effects observed:
not specified

Table 1. Results of the haematological and biochemical examination.

Dose group

HAT

leucocyte

lymphocyte

thrombocyte

GOT

GPT

Na

Cl

Ca

bilirubin

protein

cholesterol

 [mg/kg bw/day]

Males

100

-

-

-

-

-

-

-

500

-

-

-

-

-

-

1000

-

-

 

-

-

-

-

 

Females

100

-

-

-

-

-

-

-

-

-

-

-

500

-

-

-

-

-

-

-

-

-

1000

-

-

-

-

-

-

-

-

-

 /↓:slight increase/reduction

Table 2. Results of absolute/(relative) organ weight examination.

Dose group

thyroid gland

brain

adrenal gland

 [mg/kg bw/day]

males

100

-

-

500

-

- /(↓)

1000

-

-

 

females

500

-

-

 /↓:slight increase/reduction

Table 3. Results of histopathological examination.Dilation of lymph vessels in the duodenum and jenunum (number of animals with findings and grading/total number of animals in group).

Main group

Duodenum

Jejunum

 [mg/kg bw/day]

 

control

Male

0/5

Female

0/5

Male

0/5

Female

0/5

100

0/5

0/5

0/5

0/5

500

0/5

1+/5

5+/5

4+/5

1000

0/5

2+/5

0, 1+, 3++, 1+++/5

5+/5

Satellite group

Duodenum

Jejunum

 [mg/kg bw/day]

 

 

Male

Female

Male

Female

500

1+/5

3+/5

3+, 1++/5

2+/5

+,++,+++: slight, moderate, severe findings

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
read-across source
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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
Remarks:
Doses / Concentrations:
0.62, 1.25, 2.50, 5.00, 10.0 % (w/w)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
404, 809, 1583, 3067 and 5835 mg/kg bw/day
Basis:
other: actual ingested: male rats
Remarks:
Doses / Concentrations:
401, 797, 1569, 3045, 5725 mg/kg bw/day
Basis:
other: actual ingested: female rats
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
Food consumption and compound intake (if feeding study):
no effects observed
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):
no adverse biologically significant effects
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
no adverse biologically significant effects
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):
no adverse biologically significant effects
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
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 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.

HAEMATOLOGY
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 CHEMISTRY
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.

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

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. Histopathologic examination revealed an absence of compound-related lesions in any organ or tissue of rats exposed to castor oil in the diet.
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:
not specified
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
read-across source
Species:
rat
Strain:
other: Crl:WI (Han) (outbred, SPF-Quality)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277 - 353 g (males) and 183 - 225 g (females)
- Housing: 5 animals of the same sex per cage in Macrolon cages (MIV type). Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, UK) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

OTHER
- This species and strain of rat has been recognised as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 h prior to dosing and were homogenised to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE:
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

DOSE VOLUME:
5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 h.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 5 females (main study)
5 males and 5 females (recovery group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 10-day dose range finding study.
- Post-exposure recovery period in satellite groups: 14 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were conducted during the treatment phase only. Arena observations were not performed when the animals were mating, or housed individually. The time of onset, degree and duration was recorded. All symptoms were recorded and graded according to fixed scales:
Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate, grade 3 = severe, grade 4 = very severe

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION: Yes
Weekly, for males and females. Food consumption was not recorded during the mating period (except for males). Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: White blood cells, Differential leucocyte count, neutrophils, lymphocytes, monocytes, eosinophils, basophils, Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets, Prothrombin time, Activated Partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes, overnight
- How many animals: Blood samples were collected from the first 5 Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group. In addition to blood collection prior to necropsy, blood samples were collected from the Recovery animals at the end of treatment.
- Parameters checked: ALAT, ASAT, ALP, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate, Bile acids.

NEUROBEHAVIOURAL EXAMINATION: Yes
The following tests were performed on the first Main males (randomly selected at allocation), all Recovery animals and the 5 Main females from each group:
- hearing ability, pupillary reflex, static righting reflex and grip strength (score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period: 1-hour for individual animals, using a computerized monitoring system; Pearson Technical Services, Suffolk, Great Britain).
During the motor activity test, animals were caged individually. The assigned animals were tested during week 4 of treatment (all before blood sampling). Since no treatment-related findings were noted, the functional observation tests and motor activity measurements were not extended to all animals at the end of the recovery phase.

ORGAN WEIGHTS:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:

- From the first 5 Main males (randomly selected at allocation), the 5 Main females and all
Recovery animals per group: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus, Ovaries, Uterus (including cervix), Prostate, Seminal vesicles including coagulating glands, Thyroid including parathyroid.

- From all remaining males: Epididymides, Testes

Since no toxicologically relevant effect was noted on organ weights of Main females, no organ weights were collected from Repro females.
Sacrifice and pathology:
DAY OF NERCROPSY:
- Main animals: Following completion of a minimum of 28 days of dose administration.
- Recovery animals: Following completion of a minimum of 28 days of dose administration and a recovery period of 14 days.
One animal was euthanised in extremis.

GROSS PATHOLOGY: Yes
All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY: Yes
From the first 5 Main animals/sex/group, all Recovery animals, the selected Repro females/group (with live offspring) and animal no.117 that was killed in extremis (Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination):
Adrenal glands, (Aorta), Brain (cerebellum, mid-brain, cortex), Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes (including optic nerve and Harderian gland), Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, (Lacrimal gland, exorbital), (Larynx), Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), (Oesophagus), Ovaries, (Pancreas), Peyer's patches (jejunum, ileum) if detectable, Pituitary gland, Preputial gland, Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles including coagulating gland, Skeletal muscle, (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes1, Thymus, Thyroid including parathyroid (if detectable), (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

From all remaining animals and females which failed to deliver: Cervix, Clitoral gland, Coagulation gland, Epididymides, Ovaries, Preputial gland, Prostate gland, Seminal vesicles, Testes, Uterus, Vagina, All gross lesions.











Statistics:
The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
An attempt was made to transform the number of corpora lutea by using 1/x, log x, x² and √x.
However, a normal distribution was not obtained. Therefore, the number of corpora lutea was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine inter-group differences, followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
No statistical analysis was performed on histopathology findings.
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: higher body weight gain during recovery phase (males, non adverse); 100 mg/kg bw: higher body weight gain over treatment Days 8-22 (Main females; non adverse)
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: lower prothrombin time (males, non adverse); 300 mg/kg bw: lower relative lymphocyte counts (females, non adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
300, 1000 mg/kg bw: lower alanine aminotransferase; 100 mg/kg bw: higher chloride leves (males, non adverse); 1000 mg/kg bw: lower albumin and higher glucose levels (females, non adverse); higher inorganic phosphate levels (males, non adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw: lower high sensor counts (males, non adverse); 1000 mg/kg bw: effect on high sensor counts (females, non adverse)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
no adverse effects (see "Details on results")
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS
No clinical signs of toxicity were noted that were attributable to treatment.

MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. Female no. 117 (1000 mg/kg bw/day) was killed in extremis on Day 17 post-coitum. Microscopic examination revealed a marked granuloma in the bronchus region containing macrophages surrounding food particles and with central areas of necrosis. These findings were indicative of a gavage accident.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day.
Any statistically significant changes in body weight gain observed among males and females during the treatment or recovery period were considered to be of no toxicological relevance since the changes occurred in the absence of a dose-related trend and/or were of a slight and/or temporary nature. These changes consisted of a higher body weight gain for males at 1000 mg/kg bw/day during the recovery phase, a lower body weight gain of Repro females at 1000 mg/kg bw/day on Day 11 of the post coitum period, and a higher body weight gain of Main females at 100 mg/kg bw/day over treatment Days 8-22.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time in males at 1000 mg/kg bw/day and lower relative lymphocyte counts in females at 300 mg/kg bw/day at the end of treatment were considered to be of no toxicological relevance. These changes were absent at the end of the recovery period, occurred in the absence of a (clear) treatment-related trend and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. At the end of treatment these changes consisted of lower alanine aminotransferase in males at 300 and 1000 mg/kg bw/day, and higher chloride levels in males at 100 mg/kg bw/day. Changes at 1000 mg/kg bw/day at the end of the recovery phase included lower albumin and higher glucose levels in females, and higher inorganic phosphate levels in males.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
The variation in motor activity did not indicate a relation with treatment. Males at 300 mg/kg bw/day had significantly lower high sensor counts. Since the range of high sensor values encountered at 300 mg/kg bw/day was similar to that observed in the control group and no dose-related trend was noted, no toxicological relevance was ascribed to this variation. A notable variation in high sensor counts was recorded for females at 1000 mg/kg bw/day. Since the range of values at this dose was essentially similar to that observed in the control group, it was considered that no toxicologically significant effect on high sensor counts had occurred in females at 1000 mg/kg bw/day.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight
ratios.
Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a (clear) treatment-related trend, remained within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. Also, no histopathological correlates were noted to support these changes. These changes consisted of a lower spleen to body weight ratio in males at 1000 mg/kg bw/day at the end of the recovery phase, a higher spleen weight and spleen to body weight ratio in females at 100 mg/kg bw/day and the end of treatment, lower heart weight in females at 1000 mg/kg bw/day, higher heart to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment, lower adrenal weight and/or adrenal to body weight ratio in females at 300 and 1000 mg/kg bw/day at the end of treatment, and a lower ovary and ovary to body weight ratio in females at 1000 mg/kg bw/day at the end of treatment. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any toxicologically relevant alterations.
The female at 1000 mg/kg bw/day euthanized in extremis (no. 117) showed a hard, greenish nodule on the right medial lobe of the lung, dark red discolouration of the left ovary, enlarged adrenal glands, reduced size of the thymus, enlargement and greenish discolouration of the bronchial lymph node and pleura grown together with the lungs. A watery-clear cyst was found for a single control Repro female (no. 89). This finding was corroborated with a cyst found in the cervix found upon histopathological examination, which likely contributed to this animal’s suspected infertility.
Other incidental findings among control and treated animals at the end of the treatment and/or recovery period included alopecia, red foci on the thymus, reddish discolouration of the thymus or mesenteric lymph node, pelvic dilation of the kidney, reduced size of the testes, epididymides or seminal vesicles, yellowish hard nodules, a red-brown focus or tan discolouration of the clitoral glands, and fluid in the uterus. The incidence of these findings remained within the background range of findings that are encountered among rats of this age and strain, and their incidence did not show a dose-related trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment-related microscopic findings.
One control female (no. 89) had a marked cyst present in the cervix which correlated to the macroscopic finding in this animal and likely accounted for the infertility. One male rat at 100 mg/kg bw/day (no. 18) had an extensive bilateral seminiferous tubular atrophy in the testes with a subsequent extensive epididymal oligospermia, which accounted for its infertility. This was considered to be a spontaneous abnormality with no likely relationship to the test item. No other abnormalities were seen in the reproductive organs of the remaining suspected nonfertile animals which could account for their infertility. All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age and strain. The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.
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: clinical signs, functional observations, body weights, food consumption, clinical pathology, macroscopy, organ weights, and histopathology.
Key result
Critical effects observed:
not specified
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to category justification report provided in IUCLID section 13
Reason / purpose:
read-across source
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River WIGA, Sulzfeld, Germany.
- Age at study initiation: 4 weeks
- Weight at study initiation: 52-69 g (males) and 54-67 g (females)
- Housing: animals were housed in groups of 2-3 animals in Makrolon type III cages on soft wood bedding
- Diet: Altromin 1324 DK (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23
- Humidity (%): 59-67.5
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 Nov 1984 To: 19 Dec 1984
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared freshly on the day of application. The test substance was grind with a mortar before adding peanut oil.

VEHICLE
- Purity: DAB 7
- Amount of vehicle: 5 mL/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 5 days/week
Remarks:
Doses / Concentrations:
100, 500 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
10 (main study)
5 (satellite low and mid-dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Yielding of a high security range.
- Post-exposure recovery period in satellite groups: 33 days
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: on Day 28
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 28
- How many animals: all animals
- Parameters checked: erythrocytes, haematocrit, haemoglobin concentration, mean cell volume, leukocyte count, thrombocyte count and differential leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 28
- How many animals: all animals
- Parameters checked: urea, creatinine, sodium, potassium, glucose, calcium, alkaline phosphatase, glutamate oxalacetate transaminase, glutamate pyruvate transaminase, gamma-glutamyl transferase, cholesterol, total protein, chloride, bilirubin

ORGAN WEIGHTS:
- Thyroid gland, adrenal gland, thymus, spleen, heart, kidneys, brain, testes, liver
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals. Liver, thyroid gland, kidneys, adrenals, testes, uterus, ovaries, thymus, spleen, brain and heart were examined.
HISTOPATHOLOGY: Yes, from animals of vehicle control and highest dose group: Aorta thoracica, eyes, small and large intestine, glandular stomach, brain, urinary bladder, skin, heart, testes, liver, trachea, lung, maxillary lymph nodes, mesenterial lymph nodes, spleen, epididymis, adrenal gland, peripheral nerv, kidneys, ovary, pancreas, prostate, vesicular gland, thyroid gland, salivary gland, oesophagus, sceletal muscle, thymus, uterus, stomach, tongue. Target organs of digestive tract were histopathologically analysed from all animal groups.
Statistics:
t-test, U-test
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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
high-dose: reduction of HT-value (m), reduction of stab leucocytes (f); mid-dose: reduction of HT-value and reduction of leucocyte count (m), low-dose: increase of thrombocyte and leucocyte count (m), increase leucocyte count (f); non-adverse
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 1 under "Any other information on results incl. tables".
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see Table 2 under "Any other information on results incl. tables".
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see Table 3 under "Any other information on results incl. tables".
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
All animals survived the foreseen number of applications without substance-related symptoms. There were a few individual observations, which appeared at different phases of the study and were considered to be not treatment-related: one male in the control group and one female in the high-dose group showed alopecia, one female in the mid-dose group showed chromodacryorrhoea.
One male in the high-dose group died during narcosis for blood sampling at the end of the exposure period.

BODY WEIGHT AND WEIGHT GAIN
No effects were observed in group mean body weight (gain) over the whole study period.

FOOD CONSUMPTION
During the first week, a slight but statistically significant decrease in mean group food intake was observed in females of the low-dose group compared to the control group (17 vs. 18 g/rat/day), which was considered to be not toxicologically relevant. No further effects were observed in mean group food intake.

WATER CONSUMPTION
No effects were observed in group mean water intake over the whole study period.

OPHTHALMOSCOPIC EXAMINATION
No effects were observed.

HAEMATOLOGY
Low-dose males showed a slight increase in thrombocytes and lymphocytes; low-dose females showed a slight increase in leukocytes. A slight decrease in haematocrit and leukocytes and a slight increase in lymphocytes were observed in males of the mid-dose group; no effects were observed in mid-dose females. In the high-dose group, haematocrit and rod-shaped leukocytes were slightly decreased in males, and leukocytes were slightly increased in females.
Overall, haematological parameters, group and individual values did not allow the identification of a substance-related toxic effect.

CLINICAL CHEMISTRY
In low-dose males, GOT and GPT values were decreased and bilirubin values were increased; no effects on females were observed. Males in the mid-dose group showed a slight decrease in GOT and GPT as well as a decrease in calcium, while females presented a slight increase in protein and cholesterol and slight decrease in chloride. In males of the high-dose group, there was a decrease in GOT, an increase in sodium without concomitant increase in chloride, and a slight decrease in bilirubin. High-dose females showed a slight decrease in GOT and a decrease in chloride.
Overall, none of the above mentioned changes in biochemical parameters could be attributed to the test substance, since there was no dose-dependency and individual values were (with a few not dose-dependent exceptions) within the normal ranges for this species and age.

ORGAN WEIGHTS
Absolute organ weights:
Males in the low-dose group showed a slight decrease in adrenals weight. No effects were observed in low-dose females. In the mid-dose group, males showed a slight decrease in thymus weight and females showed a slight increase in brain weight. A slight decrease in thymus weight was also observed in high-dose males. High-dose females showed no effects in absolute organ weights.
Relative organ weights:
There was only a slight decrease in thymus weight in the mid-dose males.
Overall, no substance- and dose-dependent effects were observed.

GROSS PATHOLOGY
There were no substance- and dose-dependent findings. Sporadic findings included hydrometra, hydronephrosis, kidney cysts, slight redness of the stomach mucosa and inflammation of the forestomach mucosa.

HISTOPATHOLOGY: NON-NEOPLASTIC
Sporadic findings across control and treatment groups included slight interstitial inflammation in the lung, prostate inflammation, slight changes in the forestomach mucosa and increased haematopoiesis in the liver parenchyma.
In the mid- and high-dose groups, non-inflammatory dilatation of lymph vessels in the small intestine as well as foreign body giant cells and vacuolization were found in Peyer Plaques. This observed dilation of lymph vessels and foreign body giant cells (fused macrophages) in the small intestine can be interpreted as adaptive responses to a high dose of triglycerides, as the upper intestinal tract is responsible for triglyceride resorption. The histopathological findings indicated an incomplete enteral resorption. Other parts of the gastrointestinal tracts including the mesenteric lymph nodes were without treatment-related findings.
No other treatment related abnormalities were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects
Key result
Critical effects observed:
not specified

Table 1. Results of the haematological and biochemical examination.

Dose group

HAT

leucocyte

lymphocyte

thrombocyte

GOT

GPT

Na

Cl

Ca

bilirubin

protein

cholesterol

 [mg/kg bw/day]

Males

100

-

-

-

-

-

-

-

500

-

-

-

-

-

-

1000

-

-

 

-

-

-

-

 

Females

100

-

-

-

-

-

-

-

-

-

-

-

500

-

-

-

-

-

-

-

-

-

1000

-

-

-

-

-

-

-

-

-

 /↓:slight increase/reduction

Table 2. Results of absolute/(relative) organ weight examination.

Dose group

thyroid gland

brain

adrenal gland

 [mg/kg bw/day]

males

100

-

-

500

-

- /(↓)

1000

-

-

 

females

500

-

-

 /↓:slight increase/reduction

Table 3. Results of histopathological examination.Dilation of lymph vessels in the duodenum and jenunum (number of animals with findings and grading/total number of animals in group).

Main group

Duodenum

Jejunum

 [mg/kg bw/day]

 

control

Male

0/5

Female

0/5

Male

0/5

Female

0/5

100

0/5

0/5

0/5

0/5

500

0/5

1+/5

5+/5

4+/5

1000

0/5

2+/5

0, 1+, 3++, 1+++/5

5+/5

Satellite group

Duodenum

Jejunum

 [mg/kg bw/day]

 

 

Male

Female

Male

Female

500

1+/5

3+/5

3+, 1++/5

2+/5

+,++,+++: slight, moderate, severe findings

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1966
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study is reasonably well documented, meets generally accepted scientific principles and is acceptable as supporting data for limited parameters assessed.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were treated via the diet for 10 days and a limited number of parameters were evaluated. Two treatment levels of [trade name] and equivalent of its constituents were compared with two standard groups given supplements of lard, and with a control group maintained on basal ration only. The method of assessment was essentially that described by Rice et al (J. Nutrit. 61, 253, 1957), with modifications suggested by workers in the United States Food and Drug Administration.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Benger Laboratories, Holmes Chapel, England
- Weight at study initiation: 53 - 78 g
- Housing: caged individually
- Diet: after acclimatisation to a powdered diet (Spiller’s Laboratory Small Animals Diet), each rat was provided with 5 g/day of basal diet calculated to produce stable bodyweight conditions.
- Water: tap water, ad libitum


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- Mixing appropriate amounts with (Type of food): Mixture A (2 kg mix) contains beef tallow (1320 g), glycerol (290 g) and citric acid (390 g).
Basal diet (30 kg mix) contains sucrose (13000 g), casein (12000 g), cysteine (90 g), vitamin mix (1800 g), Dunn’s salt mix (1800 g), corn oil (780 g) and ‘solvitax’ cod liver oil (720 g)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
10 days
Frequency of treatment:
continuous in diet
Remarks:
Doses / Concentrations:
23.1 and 37.5%
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
23100 and 37500 mg/kg bw/day
Basis:
other: assuming a mean body weight of 80 g and a mean food intake of 8 g/day over the study period
No. of animals per sex per dose:
10 (treated) and 10 (controls)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the test was carried out in the light of a preliminary tolerance trail which indicated that:
-30%, but not 50%, test item is tolerated by young rats and affords energy and
-5%, but not 10%, citric acid is tolerated.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0, 2, 4, 6 , 9 and 11.

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
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes. The following organs were preserved routinely in buffered 4% formaldehyde prior to histopathological examination: liver, kidneys, heart, spleen, gonads, brain, thyroid and gastro-intestinal tract. Tissues were fixed for about four weeks, vacuum embedded in paraffin wax at 56°C, and sections cut at 5 µ. The latter were stained with haematoxylin and eosin.
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):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No signs of systemic toxicity and no deaths occured.

BODY WEIGHT AND WEIGHT GAIN
No biological significant differences in the body weight gain of treated and control animals occured.

ORGAN WEIGHTS
No biological significant differences between treated and control groups observed.

GROSS PATHOLOGY
No abnormalities observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No abnormalities observed.

Key result
Dose descriptor:
NOAEL
Effect level:
>= 375 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of abnormalities in clinical signs, body weight increase, organ weight, gross pathology and histopathology. Corresponding to 375000 ppm in diet assuming a mean body weight of 80 g and a mean food intake of 8 g/day for young rats.
Key result
Critical effects observed:
not specified

Table 2: Group mean bodyweight (g/rat)

Group

Weight at Day

Body weight gain

0

2

4

6

9

11

Male

 

 

 

 

 

 

 

23.1% test item

68

71

76

82

84

89

21

37.5 test item

68

72

77

83

84

90

22

23.1% Mixture A

68

72

73

80

85

89

21

37.5 % Mixture A

68

75

77

85

85

90

22

Control (basal diet)

68

67

66

70

71

75

7

Female

23.1% test item

71

74

78

82

86

90

19

37.5 test item

71

75

78

87

88

92

21

23.1% Mixture A

71

75

76

84

86

90

19

37.5 % Mixture A

70

78

79

85

84

88

18

Control (basal diet)

70

69

69

73

74

77

7

Table 3: Group mean organ weights (g)

Group

Body wt.

Liver

Kidneys

Testes

Thyroid (mg)

L & R

L & R

Male

23.1% test item

88

4.74

1.04

0.85

18

37.5 test item

89

4.63

1.11

0.93

18

23.1% Mixture A

89

4.40

0.99

1.05

20

37.5 % Mixture A

91

4.38

1.04

0.85

17

Control (basal diet)

75

3.39

0.95

0.58

14

Female

23.1% test item

88

4.14

0.92

34

21

37.5 test item

92

4.10

1.02

28

14

23.1% Mixture A

90

4.00

0.97

26

17

37.5 % Mixture A

89

4.69

1.04

32

18

Control (basal diet)

77

3.97

0.88

40

13

Table 4: Group mean organ weights as percentage of bodyweight (x100)

Group

Body wt.

Liver

Kidneys

Testes

Thyroid (mg)

L & R

L & R

Male

23.1% test item

88

536

118

96

2.08

37.5 test item

89

522

125

105

2.07

23.1% Mixture A

89

497

112

119

2.24

37.5 % Mixture A

91

482

115

94

1.88

Control (basal diet)

75

493

127

78 

1.88

Female

23.1% test item

88

469

104

3.87

2.36

37.5 test item

92

445

111

3.03

1.54

23.1% Mixture A

90

444

108

2.89

1.89

37.5 % Mixture A

89

525

117

3.59

2.03

Control (basal diet)

77

515

114

5.13

1.66

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) studies from reference substances 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 the endpoint discussion for further details). Taken together, the information from these independent sources is consistent and provides sufficient weight of evidence for hazard assessment leading to an endpoint conclusion in accordance with Annex XI, 1.2, of Regulation (EC) No 1907/2006. Therefore, the available information as a whole is sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, 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

Data on the repeated dose toxicity of glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) is limited to a 10-day feeding study only. The assessment was therefore based on the study with the registered (target) substance and with studies conducted with analogue 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).

CAS 91052-13-0

The subacute oral toxicity of Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates was investigated in Crl:WI (Han) rats according to OECD guideline 422 and in conformity with GLP (Otterdijk, 2010). Dilutions of the test substance in polyethylene glycol were administered once daily to groups of 10 male and 5 female rats at doses of 100, 300 and 1000 mg/kg bw/day for 28 days via gavage. A similar constituted group received the vehicle and served as control. In addition, satellite groups of 5 males and 5 females each for the control and high dose group were used to investigate reversibility of effects during a 14-day post-exposure recovery period. No substance-related mortalities and clinical signs occurred during the whole study period. All parameters assessed at neurobehavioural examination were found to be normal and comparable to controls. Food consumption was similar between treated and control animals and no toxicologically relevant changes in body weights and body weight gain were noted up to and including 1000 mg/kg bw/day. Any statistically significant changes in clinical biochemistry and haematology parameters were considered to be of no toxicological relevance as these occurred in the absence of a clear treatment-related trend and remained within the range considered normal for rats of this age and strain. Any statistically significant changes in organ weights and organ to body weight ratios were considered to be of no toxicological relevance as these occurred in the absence of a clear treatment-related trend. Further, organ weight changes were within the range considered normal for rats of this age and strain and/or were present at the end of the recovery period only. In addition, no histopathological correlates were noted which could support organ weight changes. At necropsy, all incidental findings remained within the background range of findings that are encountered among rats of this age and strain. Since no dose-related trend was observed, these findings were considered to be of no toxicological relevance. Based on the results of this subacute toxicity study, the NOAEL for Crl:WI rats was considered to be ≥ 1000 mg/kg bw/day.

CAS 91845-19-1

To assess the subacute toxicity of Glycerides, C16-18 and C18-hydroxy mono- and di-, a GLP-conform study according to EU method B.7 was performed in Sprague Dawley rats at doses of 100, 500 and 1000 mg/kg bw/day (Potokar, 1985). The test substance in peanut oil or the vehicle alone was administered once daily to groups of 10 animals per sex via gavage for a period of 28 days. A satellite group of 5 males and 5 males, each for the control and 500 mg/kg bw/day dose groups, was included in the study to investigate reversibility of effects during a 33-day post-exposure recovery period. No mortality and no clinical signs were observed during the whole study period. At ophthalmological examination no adverse effects were reported. Body weight gain and food consumption were not altered between treated and control groups. Haematology and clinical chemistry analysis did not reveal any treatment-related changes in the animals. At macroscopic examination of the animals, no abnormal findings were observed. Histopathological examination of animals treated with 500 and 1000 mg/kg bw/day revealed non-inflammatory dilatation of lymph vessels in the small intestine as well as foreign body giant cells and vacuolization in Peyer Plaques. However, these findings were considered as adaptive responses to treatment, but not as adverse effects. Based on the study results, a NOAEL of ≥ 1000 mg/kg bw/day was set for male and female Sprague Dawley rats.

CAS 8001-79-4

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 (NTP, 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.

CAS 91052-16-3

A 10-day feeding study which was carried out to compare the nutritive value of glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) with that of its constituents beef tallow, glycerol and citric acid is available. The study is reasonably well documented, meets generally accepted scientific principles and is acceptable as supporting data for the limited parameters assessed. Sprague-Dawley rats were treated with the test substance via the diet for 10 days. Two treatment levels of glycerides, C16-18 and C18-unsaturated mono-and di-citrates, 23.1% (23100 mg/kg bw/day) and 37.5% (37500 mg/kg bw/day), and equivalent level of its constituents were compared with two standard groups given supplements of lard, and with a control group maintained on basal ration only. There were 5 groups with 10 males and 10 females. The method of assessment was essentially that described by Rice et al (J. Nutrit. 61, 253, 1957), with modifications suggested by workers in the United States Food and Drug Administration. After 10 days of treatment, all animals were sacrificed by CO2 euthanasia and the following analysis was conducted: organ weights, gross pathology and histopathology. No mortalities and no signs of ill-health or abnormal behaviour was observed during the study. No test substance related effect on body weight or organ weight was observed and the gross pathology and histopathology investigations did not reveal any treatment-related findings. Based on the study results, a NOAEL of ≥ 37500 mg/kg bw/day was set for male and female Sprague Dawley rats.

 

Overall conclusion for repeated dose toxicity

The data on the registration (target) substance and on the read-across analogue substances showed that no adverse effects were observed up to and including the recommended limit value. Therefore, as the available data did not identify any hazard for repeated dose toxicity, glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) is not considered to be hazardous following repeated exposure.

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Hazard assessment is conducted by means of read-across from structural analogues. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between the source and target substances and overall quality assessment (refer to the endpoint discussion for further details).

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 glycerides, C16-18 and C18-unsaturated mono-and di-citrates (CAS 91052-16-3) data will be generated from data 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 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.