# 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 substanc e 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 (Combine d 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 

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

5/31/2012 file://G:\TSCA\Test Rule - Ox idized Castor Oil\Robust Stud y Summaries\OECD 422.html

## 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 ti me 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 

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

5/31/2012 file://G:\TSCA\Test Rule - Ox idized Castor Oil\Robust Stud y Summaries\OECD 422.html

## Control animals



# Examinations

Any other information on materi als 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 pr egnancy or following the prescribed 14-day mating period, female s 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 assign ed and paired with alternate male rats that have successfully mated. These female rats remained in cohabitation with the second se t 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. Aft er 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 cou nt

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 p rotein; 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). 

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

5/31/2012 file://G:\TSCA\Test Rule - Ox idized Castor Oil\Robust Stud y Summaries\OECD 422.html

# 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 

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

5/31/2012 file://G:\TSCA\Test Rule - Ox idized Castor Oil\Robust Stud y Summaries\OECD 422.html

# 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). (Provantis TM version 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 with p values < 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 Ox idized Castor Oil at any stage throughout the course of the study. Any changes were considered incidental and toxicologically insignif icant.

Female: There were no clinical signs in females (reproductive a nd general toxicity groups) attributable to the administration o f 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 w ere 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 o f 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. 

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

5/31/2012 file://G:\TSCA\Test Rule - Ox idized Castor Oil\Robust Stud y Summaries\OECD 422.htmlFemale: There were no changes in female body weight or body weight gain attributable to the administration of Oxidized Castor O il 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, consid ered 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 weig ht 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 significan t changes from vehicle control in any of the fertility or reproductive parameters evaluated during the course of the present stud y.

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 perfor med.

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 w ith 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 o f 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. 

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

5/31/2012 file://G:\TSCA\Test Rule - Ox idized Castor Oil\Robust Stud y Summaries\OECD 422.html

## 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). Specifica lly, there were no relevant changes in the clinical observations, number, viability, weight or sex ratio associated in any of the da ms 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 Cas tor 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. 

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

5/31/2012 file://G:\TSCA\Test Rule - Ox idized Castor Oil\Robust Stud y Summaries\OECD 422.html