Endpoint study record: Combined 28-day repeated dose oral 

in rats - PSL 2012 

Administrative Data 

Data source 

Reference 

Data access 

Data protection claimed 

Materials and methods 

Test type 

Limit test 

Test guideline 

GLP compliance 

Test materials 

Identity of test material same as for substance defined in section 1 (if not read-across) 

UUID IUC5-9fe65d61-ad9c-4fa4-96f9-bf32687da49c 

Dossier UUID 0 

Author mbogle / Vertellus Specialties Inc.

Date 2012-05-31 10:43:30 EDT 

Remarks 

 

Purpose flag key study; robust study summary; used for classification; used for MSDS 

Study result type experimental result Study period 12 December 2011 to 14 May 2012 

Reliability 1 (reliable without restriction) 

Reference 

type 

Author Year Title Bibliographic 

source 

Testing 

laboratory 

Report 

no. 

Owner 

company 

Company 

study no. 

Report 

date 

study 

report 

Bauter, 

M.R. 

2012 Oxidized Castor Oil: 

Combined 28-day repeated 

dose oral toxicity study with 

the 

reproduction/developmental 

toxicity screening test in rats 

 Product 

Safety 

Labs, 

Dayton, NJ 

USA 

33479 Vertellus 

Specialties 

Inc. 

 

data submitter is data owner 

yes, but willing to share 

combined repeated dose and reproduction / developmental screening 

no 

Qualifier Guideline Deviations 

according 

to 

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

Toxicity Screening Test) 

no 

according 

to 

other guideline: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with 

Reproduction/Developmental Toxicity Screening Test, July 2000) 

no 

according 

to 

other guideline: EPA OTS 40 CFR 799.9365 (TSCA Combined Repeated Dose Toxicity Study with the 

Reproduction/Developmental Toxicity Screening Test, July 2011) 

no 

yes 

yes 

Endpoint study record: Combined 28-day repeated dose oral in rats - PSL 2012 Page 1 of 7

file://G:\TSCA\Test Rule - Oxidized Castor Oil\Robust Study Summaries\OECD 422.html 5/31/2012
Test material identity 

Details on test material 

Test animals 

Species 

Strain 

Sex 

Administration / exposure 

Route of administration 

Vehicle 

Analytical verification of doses or concentrations 

Duration of treatment / exposure 

Frequency of treatment 

Doses/concentrations 

No. of animals per sex per dose 

Identifier Identity 

CAS name Castor Oil, Oxidized 

CAS number 68187-84-8 

- Name of test material (as cited in study report): Oxidized Castor Oil (aka: Pale 16, Material #104495)

- Physical state: clear, yellow-amber liquid with a mild odor 

- Analytical purity: 100% 

- Lot/batch No.: 0000113649 

- Expiration date of the lot/batch: 6/30/12 

- Storage condition of test material: Ambient 

rat 

other: Crl: CD®(SD) IGS rat 

male/female 

oral: gavage 

olive oil (with approximately 5% ethanol added to facilitate solubilization) 

yes 

Male Rats 

- Reproduction/General Toxicity Groups: During two week pre-mating and two week mating periods for at least 28 days of 

exposure. 

Female Rats 

- Reproduction Groups: During two week pre-mating, two week mating, gestation and lactation periods. 

- General Toxicity Groups: For at least 28 days of exposure. 

Dose Frequency: Each animal was dosed by oral intubation using a stainless steel ball-tipped gavage needle attached to an 

appropriate syringe. Dose administration was daily (7 days/week) for all adult animals. Dosing was at approximately the same time 

each day ±2 hours with an exception on the days the hematology and/or clinical chemistry samples were collected. 

Dose level 250 mg/kg/day, as dose concentration 50 mg/mL 

Basis actual ingested 

Dose level 500 mg/kg/day, as dose concentration 100 mg/mL 

Basis actual ingested 

Dose level 1000 mg/kg/day, as dose concentration 200 mg/mL 

Basis actual ingested 

Male Rats 

Endpoint study record: Combined 28-day repeated dose oral in rats - PSL 2012 Page 2 of 7

file://G:\TSCA\Test Rule - Oxidized Castor Oil\Robust Study Summaries\OECD 422.html 5/31/2012
Control animals 

Examinations 

Any other information on materials and methods incl. tables 

- Reproduction Groups (total 10 rats each): Groups 1A, 2A, 3A, and 4A.

- General Toxicity Groups (5 rats each): Groups 1B, 2B, 3B and 4B. 

Female Rats 

- Reproduction Groups (total 10 rats each): Groups 1A, 2A, 3A and 4A.

- General Toxicity Groups (5 rats each): Groups 1B, 2B, 3B and 4B. 

yes, concurrent vehicle 

Age at study initiation:9 weeks at receipt, seven days acclimation period before start of testing.

Mating procedures:Males and females were dosed while housed separately for a 14-day pre-mating period, followed by dosing 

through a 14-day co-habitation period. Upon determination of pregnancy or following the prescribed 14-day mating period, females 

were removed to a separate cage and continued to be dosed through the gestation period of pregnancy and Day 4 of lactation.

After two weeks of treatment, animals were randomly mated by placing one female in the breeding cage of a male from the same 

dose group. A record of mating pairs was maintained.

The cohabitation period consisted of a maximum of 14 days. Vaginal smears were performed daily on females during the mating 

period and the presence or absence of sperm or vaginal plug was recorded. Females considered to have successfully mated 

(determined by the presence of vaginal sperm and/or a vaginal copulation plug) were removed from the cage of the male, assigned

a GD 0 and housed individually. Female rats that had not mated within the first seven days of cohabitation were randomly assigned 

and paired with alternate male rats that have successfully mated. These female rats remained in cohabitation with the second set of 

males for a maximum of seven additional days. If successful mating had not occurred after 14 days of cohabitation, females were

assigned a GD 0 on the morning of the last day of the cohabitation period and were evaluated as if in gestation thereafter. After the 

mating period, any female with no positive evidence of mating was individually housed in a plastic solid bottom polycarbonate 

shoebox cage containing nesting material.

Clinical Pathology

Hematology:erythrocyte count; hemoglobin concentration; hematocrit; mean corpuscular volume; mean corpuscular hemoglobin; 

red cell distribution width; absolute reticulocyte count; platelet count; total white blood cell and differential leukocyte count

Coagulation:prothrombin time; active partial thromboplastin time

Clinical Chemistry:serum aspartate aminotransferase; serum alanine aminotransferase; sorbitol dehydrogenase; alkaline 

phosphatase; total bilirubin; urea nitrogen; blood creatinine; total cholesterol; triglycerides; fasting glucose; total serum protein; 

albumin; globulin; calcium; inorganic phosphorus; sodium; potassium; chloride; creatine phosphokinase; lactate dehydrogenase; 

uric acid

Urinalysis:quality; pH; ketone; color; glucose; bilirubin; clarity; specific gravity; blood; volume; protein; urobilinogen; microscopic 

urine sediment examination

Necropsy:At terminal sacrifice, all reproductive female animals were euthanized by exsanguination from the abdominal aorta under 

carbon dioxide anesthesia. All animals in the study were subjected to a necropsy, which included examination of the external 

surface of the body, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities and their contents.

- Reproductive Group (males): prostate, seminal vesicle, and coagulating gland (combined), testes (combined), epididymides 

(combined)

- Reproductive Group (females): conceptuses, corpora lutea count, non-gravid uterus, cervix and fallopian tubes. ovaries 

(combined)

- General Toxicology Groups:

z Weighed wet immediately: adrenals (combined); kidneys (combined); spleen; brain;

liver; thymus; epididymides; ovaries with oviducts; uterus with cervix; heart; testes

(combined). 

Endpoint study record: Combined 28-day repeated dose oral in rats - PSL 2012 Page 3 of 7

file://G:\TSCA\Test Rule - Oxidized Castor Oil\Robust Study Summaries\OECD 422.html 5/31/2012
Results and discussions 

Effect levels 

Results of examinations 

Any other information on results incl. tables 

z Preserved in 10% neutral buffered formalin for possible future histopathological

examination: seminal vesicles (with coagulating glands); jejunum; skeletal muscle;

kidneys; skin; larynx; spinal cord ‐ 3 levels: cervical, mid‐ thoracic, and lumbar; adrenals;

liver; all gross lesions; lungs; aorta; mammary gland; spleen; bone (femur); nose & nasal

turbinates; sternum; brain – 3 sections cerebellum, cerebrum, medulla/pons; ovaries &

oviducts; stomach; pancreas; thymus; thyroid/parathyroid; peripheral nerve (sciatic);

trachea; cecum; pharynx; urinary bladder; colon; pituitary; uterus with cervix;

duodenum; rectum; vagina; esophagus; lymph node mesenteric; prostate; Harderian

gland; lymph node mandibular; heart; bone marrow collected with femur & sternum;

ileum with peyer’s patches; salivary glands (sublingual, submandibular, parotid)

z Preserved in modified Davidson’s fixative and then stored in ethanol for possible future

histopathological examination:epididymides; eyes with optic nerve; testes

z Histopathology: Histopathology examination was performed on the preserved organs

and tissues of the Reproductive and General Toxicity Group animals from the control and

high dose groups (with special emphasis on stages of spermatogenesis in the male

gonads and histopathology of interstitial testicular cell structure). In addition, gross

lesions of potential toxicological significance noted in any test groups at the time of

terminal sacrifice were also examined. These examinations were extended to the spleen

of a Group 2 female (7049), the uterus of each of two Group 3 females (7071 and 7072),

and the esophagus of a Group 3 male (7055).

z Filial (F1) Generation: Examinations included clinical observations as soon as possible after delivery, and thereafter twice

daily. Individual pup weights recorded on Day 1 and Day 4 after delivery. All surviving pups were sacrificed on Day 4 by lethal

injection of sodium pentobarbital. The thorax, abdomen, and pelvis of each pup were examined internally. Internal sex was

determined and recorded for all pups. All pups were examined for abnormalities of the palate. Pups with gross lesions were retained

in 10% NBF for possible future evaluation.

Endpoint Effect 

level 

Based 

on 

Sex Basis for effect level / Remarks 

NOAEL 1000 

mg/kg 

bw/day 

(actual 

dose 

received) 

test 

mat. 

male/female In conclusion, male and female parental animals treated with 250, 500 and 1000 

mg/kg/day Oxidized Castor Oil exhibited normal parameters and comparable 

values to controls during the pre-mating, mating, post-mating (males) and 

gestational and lactational (females) phases throughout the study. Parental fertility 

and reproductive indices, including evaluation of the stages of spermatogenesis 

and interstitial testicular cell structure, revealed no test substance related effects. 

Furthermore, the filial generation from all test groups exhibited normal parameters 

in accordance with that expected for this strain of rat (CRL, 1993). Specifically, 

there were no relevant changes in the clinical observations, number, viability, 

weight or sex ratio associated in any of the dams or their litters upon 

administration of Oxidized Castor Oil at any dose level. 

Therefore, under the conditions of this study, the no-observed-adverse-effect level 

(NOAEL) for the test substance Oxidized Castor Oil, was 1000 mg/kg/day, the 

highest dosage tested, for male and female Sprague-Dawley rats in this combined 

repeated dose oral toxicity study with reproduction/developmental screening. 

STATISTICAL ANALYSIS

Endpoint study record: Combined 28-day repeated dose oral in rats - PSL 2012 Page 4 of 7

file://G:\TSCA\Test Rule - Oxidized Castor Oil\Robust Study Summaries\OECD 422.html 5/31/2012
Overall remarks, attachments 

Overall remarks 

Significance was judged at a probability value of p < 0.05. Male and female rats were evaluated separately.

A. Statistical Methods: In‐Life and Organ Weight Data (P):

Mean and standard deviations were calculated for all quantitative data. If warranted by sufficient group sizes, data within groups were

evaluated for homogeneity of variances and normality by Bartlett’s test (Bartlett, 1937). Where Bartlett’s test indicates homogeneous

variances, treated and control groups were compared using a one‐way analysis of variance (ANOVA). When oneway analysis of variance was

significant, a comparison of the treated groups to control by Dunnett’s test (Dunnett, 1964, 1980) for multiple comparisons was performed.

Where variances are considered significantly different by Bartlett’s test, groups were compared using a nonparametric method (Kruskal‐Wallis

non‐parametric analysis of variance; Kruskal‐Wallis, 1952). When non‐parametric analysis of variance was significant, comparison of treated

groups to control was performed using Dunn’s test (Dunn, 1964). (ProvantisTMversion 8.4.2.0, Tables and

Statistics, Instem LSS, Staffordshire UK; INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Motor activity data was further evaluated by

two‐way repeated measures analysis.

B. Statistical Methods (Clinical Pathology)

Test substance dose‐groups were compared to the control group. Continuous data (e.g., clinical chemistry, hematology, etc.) that were

examined statistically may be evaluated for equality or homogeneity of variance using Bartlett's Test (Bartlett, 1937). Group means are to be

analyzed by a one‐way variance analysis (ANOVA) followed by Dunnett's Test (Dunnett, 1964, 1980). Alternatively, continuous data (e.g.,

clinical chemistry, hematology, etc.) determined to be nonhomogeneous, may be analyzed using a Kruskal‐Wallis Analysis of Variance Kruskal‐

Wallis, 1952) followed by a pairwise Mann‐Whitney U Test (Mann and Whitney, 1947). For the Bartlett Test, a probability (p) level < 0.001 will

be considered significant. For all other statistical tests, differences withpvalues < 0.05 were considered statistically significant.

C. Statistical Methods of the Filial (F1) Generation:

The litter was the experimental unit for evaluation where appropriate. Mean and standard deviation were calculated independently for all test

groups. Similarly, mortality and sex was analyzed as number per litter. These analyses were performed independently on all test groups with no

inferential comparisons being made with regards to homogeneity of variance, normality, and analysis of variance.

Test Substance and Dose Preparation Analysis:Based on the stability, homogeneity, and dose concentration verification results, 

the animals are considered to have received the targeted dose concentrations of Oxidized Castor Oil.

Parental Generation (P)

In-Life Evaluations

Mortality and Clinical Observations

There were no test substance-related or other mortalities of the parental animals during the course of the study.

Male: There were no clinical signs in males (reproductive and general toxicity groups) attributable to the administration of Oxidized 

Castor Oil at any stage throughout the course of the study. Any changes were considered incidental and toxicologically insignificant.

Female: There were no clinical signs in females (reproductive and general toxicity groups) attributable to the administration of 

Oxidized Castor Oil at any stage throughout the course of the study. Any changes were considered incidental and toxicologically

insignificant.

Functional Observational Battery

In general, the functional behavioral results of the general toxicity test groups of male and female rats performed on Day 35 were 

considered comparable to the control groups. Any decreases in quantitative measurements or increases in incidence of open field

measurements were minimal and not associated with a constellation of findings that would support a toxicologically significant 

behavioral change.

Motor Activity

The Motor Activity results (i.e., mean Total Movements) of the test groups of male and female rats performed on Day 33 were 

considered comparable to the control groups. In general, all groups of animals (including controls) exhibited a similar level of 

movement over all intervals. No statistically significant differences were noted in any male or female test groups compared to their 

corresponding control groups.

Body Weight and Body Weight Gain

Male: There were no changes in male body weight or body weight gain attributable to the administration of Oxidized Castor Oil 

compared with control values.

Endpoint study record: Combined 28-day repeated dose oral in rats - PSL 2012 Page 5 of 7

file://G:\TSCA\Test Rule - Oxidized Castor Oil\Robust Study Summaries\OECD 422.html 5/31/2012
Female: There were no changes in female body weight or body weight gain attributable to the administration of Oxidized Castor Oil 

compared with control values. Any changes were considered incidental and toxicologically insignificant.

Food Consumption and Food Efficiency

Male: Mean daily food consumption and food efficiency for males administered Oxidized Castor Oil remained comparable to control

throughout the study.

Female: Mean daily food consumption and food efficiency for females administered Oxidized Castor Oil remained comparable to 

control for all of the reproductive and general toxicity groups throughout the study.

Post-Mortem Study Evaluations

Clinical Pathology

Clinical pathology performed on blood samples of 5 males and females each in the general toxicity groups revealed no test 

substance-related changes in hematology, coagulation, clinical chemistry or urinalysis. In summary, under the conditions of the

present study, there were no hematologic, clinical chemistry, coagulation or urinalysis changes in male and female rats, considered 

related to the administration of Oxidized Castor Oil. Any changes were considered incidental and non-adverse as they were not 

accompanied by any other clinical or histological change. As such, these and all other changes were considered toxicologically 

insignificant.

Sacrifice and Gross Necropsy

Mortality:All adult animals survived to scheduled necropsy.

Macroscopic Findings:At scheduled sacrifice Day 36/37 (general toxicity/reproductive males on Day 36 and general toxicity 

females on Day 37) or LD 4 (reproductive females), there were no macroscopic findings related to the administration of the test

substance, Oxidized Castor Oil.

Microscopic Findings:Histopathologic evaluation of the Group 1 and 4 males (reproductive and general toxicity) and females 

(general toxicity) revealed no microscopic findings related to the administration of the test substance, Oxidized Castor Oil.

Organ Weight and Organ Weight Ratios:All organ weights for the adult treated reproductive and general toxicity animals at 250, 

500, and 1,000 mg/kg/day of Oxidized Castor Oil appeared to be within normal ranges for the protocol-directed tissues for this age 

and strain of rat with respect to sex compared with control (Sugimoto et al, 2000). Similarly, no remarkable organ-to-body weight 

changes were observed compared with control values. There were no differences in absolute or relative organ weights between 

groups.

Reproductive Performance

Oxidized Castor Oil, when administered to parental rats during a four weeks premating and mating period, produced no significant 

changes from vehicle control in any of the fertility or reproductive parameters evaluated during the course of the present study.

Offspring Generation (F1)

Individual F1 Fate: There were no F1 clinical findings or congenital anomalies deemed related to the administration of the test

substance, Oxidized Castor Oil. One Group 3 pup (7068-13) of indeterminate sex was sacrificed humanely soon after birth on LD 0

with imperforate anus and anury (Group 3 malformation incidence: 0.7%), presumably incompatable with survival. This finding was

considered incidental and of no toxicologic significance since it is a singular occurrence and no further evaluation was performed.

Several pups were found dead, euthanized, or missing on LD 0-4. Three of 132 (2.3%), 1/141 (0.7%), 4/135 (3.0%), and 3/131 

(2.3%) in Groups 1-4, respectively. One pup was found miss-sexed between LD 0-4 (0.1%).

Clinical Observations (F1):All surviving pups appeared normal during the study, with the exception of the following pups: One pup 

from Group 1 (litter 7012) on LD 2 – LD 4 was missing a tail tip, and one pup from Group 3 (litter 7068) on LD 0 was observed with 

absent tail (anury) and imperforate anus; one pup from Group 2 (litter 7039) had bruising on LD 1. All pups were observed with milk 

bands except for one pup from Group 4 (litter 7095) on LD 4.

Body Weights and Body Weight Gain (F1):All litters increased in weight during the lactation phase. For the LD 1-LD 4 period, 

Group 1 litters had an average gain of 31.73 grams corresponding to a 36.68% weight gain, Group 2 litters had an average gain of 

31.87 grams for a 34.31% weight gain, Group 3, 27.72 grams for a 31.78% gain, and Group 4, 26.98 grams for a 32.27% gain. 

There were no statistical differences observed in treated groups when compared to controls.

Endpoint study record: Combined 28-day repeated dose oral in rats - PSL 2012 Page 6 of 7

file://G:\TSCA\Test Rule - Oxidized Castor Oil\Robust Study Summaries\OECD 422.html 5/31/2012
Attached full study report 

Applicant's summary and conclusion 

Conclusions 

Executive summary 

Necropsy Observations (F1):All surviving pups were observed with no abnormalities at necropsy with the exception of one pup 

from Group 3 (litter 7068) observed with absent tail (anury) and imperforate anus upon external exam. All pups were observed to

have milk bands present.

Attached full study report 

Oxidized Castor Oil - 28-Day Oral Toxicity in Rats - OECD 421 (PSL 2012).pdf / 12.47 MB (application/pdf) 

NOAEL for Oxidized Castor Oil was 1000 mg/kg/day, the highest dosage tested, for male and female Sprague-Dawley rats in a 

combined repeat dose/repro/developmental screening assay. 

In conclusion, male and female parental animals treated with 250, 500 and 1000 mg/kg/day Oxidized Castor Oil exhibited normal 

parameters and comparable values to controls during the pre-mating, mating, post-mating (males) and gestational and lactational

(females) phases throughout the study. Parental fertility and reproductive indices, including evaluation of the stages of 

spermatogenesis and interstitial testicular cell structure, revealed no test substancerelated effects. Furthermore, the filial generation 

from all test groups exhibited normal parameters in accordance with that expected for this strain of rat (CRL, 1993). Specifically, 

there were no relevant changes in the clinical observations, number, viability, weight or sex ratio associated in any of the dams or 

their litters upon administration of Oxidized Castor Oil at any dose level.

Therefore, under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for the test substance Oxidized Castor 

Oil, was 1000 mg/kg/day, the highest dosage tested, for male and female Sprague-Dawley rats in this combined repeated dose oral

toxicity study with reproduction/developmental screening.

Endpoint study record: Combined 28-day repeated dose oral in rats - PSL 2012 Page 7 of 7

file://G:\TSCA\Test Rule - Oxidized Castor Oil\Robust Study Summaries\OECD 422.html 5/31/2012