SRC TR

- 12

- 078

HPV 2 TEST RULE DATA ADEQUACY REVIEW ( FINAL )

Castor Oil, Oxidized; CASRN 68187

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Prepared by:

Julie Ramage, Fernando Llados, Teresa Manyin, and William L. Richards

Defense and Environmental Solutions Division SRC, Inc.

7502 Round Pond Road North Syracuse, NY 13212

Contract No. EP

- W

- 09

- 027

Task FG007. 3 .009.001

Submitted to:

OCSPP/OPPT/RAD (7403M) U.S. Environmental Protection Agency 1200 Pennsylvania Avenue

Washington, D.C. 20460

Bryan Lobar , Project Officer

Meena Sonawane, Work Assignment Manager David Brooks, Alternate Work Assignment Manager

October 24, 2012Final HPV 2 Data Adequacy Review CAS RN 68187

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i TABLE OF CONTENTS

A. SOURCE MATERIAL USED FOR ASSESSMENT

................................ .............................. 1

B. EVALUATING DATA ADEQUACY

................................ ................................ ..................... 1

C

. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST RULE

........ 1

D. DATA FOR SIDS ENDPOINTS

................................ ................................ .............................. 2

D

- 1. Acute Toxicity to Fish

................................ ................................ ................................ ....... 2

D

- 2. Acute Toxicity to Aquatic Invertebrates

................................ ................................ ............ 3

D

- 3. Toxicity to Aquatic Plants

................................ ................................ ................................ . 4

D

- 4. Acute Toxicity

................................ ................................ ................................ .................... 5

D

- 5. Repeat ed

- Dose/Reproductive/Developmental Toxicity

................................ .................... 9

D

- 6. Genetic Toxicity

– Gene Mutation

................................ ................................ ................... 17

D

- 7. Genetic Toxicity

– Chromosomal Aberrations

................................ ................................ 21

E. DATA ADEQUACY SUMMARY TABLE

................................ ................................ ........... 25Final HPV 2 Data Adequacy Review CAS RN 68187

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1 A. SOURCE MATERIAL USED FOR ASSESSMENT

This document evaluates a submission from Vertellus Specialties Inc. (dated May 31

, 2012) received by EPA in response to the final test rule, “Testing of Certain High Production Volume Chemicals; Second Group of Chemicals” (76 FR 1067, January 7, 2011; referred to herein as HPV2) for castor oil, oxidized (CASRN 68187

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- 8 ). In this submission, Vertellus Specialties Inc. sup plied data for repeated

- dose, reproductive, and developmental toxicity; gene mutation ; and chromosomal aberrations endpoints required by the HPV2 test rule. These newly submitted studies are evaluated for adequacy herein. This report also contains acute toxicity data, evaluated previously by EPA, that were derived from a prior submission by Vertellus Specialties Inc. ( dated October 22, 2008 ).

B. EVALUATING DATA ADEQUACY

Data available for SIDS toxicity endpoints are evaluated for adequacy in Section D

. Each study was summarized according to a standard format and assigned a Klimisch reliability code based on its scientific merit and conduct, using professional judgment. To determine whether data were adequate to satisfy SIDS endpoints, study details w ere compared to current standard test guidelines to establish whether the study met current testing requirements. Studies for a common endpoint are grouped together in the document and an endpoint adequacy assessment is provided near the top of the first page of the summaries for that endpoint. If the data available for the endpoint were determined to be inadequate, a basis for that conclusion is supplied. A table summarizing data availability/adequacy for each SIDS endpoint is included on the last page of this document (Section E).

C

. SUMMARY OF DATA ADEQUACY FOR TESTING REQUIRED BY TEST

RULE

D ata supplied on May 31 , 2012 by Vertellus Specialties Inc. are considered adequate to satisfy the gene mutation (E1), chromosomal aberrations (E2) , and repeated

dose/reproductive/developmental toxicity (F1) test requirement s promulgated in the final

HPV2 test rule.

There are no data gaps remaining for any SIDS endpoints.Final HPV 2 Data Adequacy Review CAS RN 68187

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2 D . DATA FOR SIDS ENDPOINTS

D

- 1

. Acute Toxicity to Fish

Summary of endpoint: No data are available for this endpoint. EPA is not requiring testing of CASRN 68187

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- 8 for aquatic toxicity endpoint s ; this testing is not considered feasible due to the extremely poor water solubility of this substance.Final HPV 2 Data Adequacy Review CAS RN 68187

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3 D

- 2

. Acute Toxicity to Aquatic Invertebrates

Summary of endpoint: No data are available for this endpoint. EPA is not requiring testing of CASRN 68187

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- 8 for aquatic toxicity endpoints; this testing is not considered feasible due to the extremely poor water solubility of th is substance.Final HPV 2 Data Adequacy Review CAS RN 68187

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4 D

- 3

. Toxicity to Aquatic Plants

Summary of endpoint: No data are available for this endpoint. EPA is not requiring testing of CASRN 68187

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- 8 for aquatic toxicity endpoints; this testing is not considered feasible due to the extremely poor water solubility of this substance.Final HPV 2 Data Adequacy Review CAS RN 68187

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5 D

- 4. Acute Toxicity

Summary of endpoint: Adequate

Study 1: Acute oral toxicity study in rats ( Vertellus Specialties Inc. 2008 )

Title: Acute Oral Toxicity Study

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor Oil, Oxidized; CASRN 68187

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Remarks: Not available (see DCN 40100000003 )

METHOD

Method/guideline followed: Not available (see DCN 40100000003)

Test type: Acute Oral

- Rat

GLP compliant?: Not available (see DCN 40100000003 )

Year study performed: Not available (see DCN 40100000003 )

Test species: Rats

Number of animals/sex/dose: Not available (see DCN 40100000003)

Route of administration: Not available (see DCN 40100000003)

Vehicle: Not available (see DCN 40100000003)

Doses/concentrations tested: Not available (see DCN 40100000003)

Statistical methods used: Not available (see DCN 40100000003)

Test method/conditions remarks: Not available (see DCN 40100000003 )

RESULTS

Endpoint value(s): LD 50 > 5000 mg/kg

- bw ( value is from Doc ument ID EPA

- HQ

OPPT

- 2007

- 0531

- 0065; see R eferences )

Results remarks: Not available (see DCN 40100000003)Final HPV 2 Data Adequacy Review CAS RN 68187

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6 CONCLUSIONS

The oral LD 50 in rats was > 5000 mg/kg

- bw.

STUDY RELIABILITY

[ 1 ] Reliable with out restrictions.

REFERENCES

Vertellus Specialties Inc. 2008. Study submitted as CBI, DCN 40090000005

. Summary provided in October 22, 2008 letter from Vertellus Speciaties, Inc. re: 73 FR 43314

– Testing of Certain High Production Volume Chemicals, Second Group of Chemicals (Oxidized Castor Oil), Document ID EPA

- HQ

- OPPT

- 2007

- 0531

- 0065

, http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0065

. A detailed, CBI data adequacy review of this study is found in DCN 40100000003.Final HPV 2 Data Adequacy Review CAS RN 68187

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7 Study 2 : Acute dermal toxicity study in rats (Vertellus Specialties Inc. 2008)

Title: Acute Dermal Toxicity Study

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor Oil, Oxidized; CASRN 68187

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Remarks: Not available (see DCN 40100000003 )

METHOD

Method/guideline followed: Not available (see DCN 40100000003)

Test type: Acute Dermal

- Rat

GLP compliant?: Not available (see DCN 40100000003 )

Year study performed: Not available (see DCN 40100000003 )

Test species: Rabbits

Number of animals/sex/dose: Not available (see DCN 40100000003)

Route of administration: Not available (see DCN 40100000003)

Vehicle: Not available (see DCN 40100000003)

Doses/concentrations tested: Not available (see DCN 40100000003)

Statistical methods used: Not available (see DCN 40100000003)

Test method/conditions remarks: Not available (see DCN 40100000003 )

RESULTS

Endpoint value(s): LD 50 > 2 000 mg/kg

- bw

Results remarks: Not available (see DCN 40100000003)

CONCLUSIONS

The dermal LD 50 in rats was > 2 000 mg/kg

- bw (value is from Doc ument ID EPA

- HQ

OPPT

- 2007

- 0531

- 0065; see R eferences )Final HPV 2 Data Adequacy Review CAS RN 68187

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8 STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCES

Vertellus Specialties Inc. 2008. Study submitted as CBI, DCN 40090000005 . Summary provided in October 22, 2008 letter from Vertellus Speciaties, Inc. re: 73 FR 43314

– Testing of Certain High Production Volume Chemicals, Second Group of Chemicals (Oxidized Castor Oil), Document ID EPA

- HQ

- OPPT

- 2007

- 0531

- 0065

, http://www.regulations.gov/#!documentDetail;D=EPA

- HQ

- OPPT

- 2007

- 0531

- 0065 . A detailed, CBI data adequacy review of this study is found in DCN 40100000003.Final HPV 2 Data Adequacy Review CAS RN 68187

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9 D

- 5. Repeated

- Dose/Reproductive/Developmental Toxicity

Summary of repeated

- dose toxicity endpoint: Adequate Summary of reproductive toxicity endpoint: Adequate Summary of developmental toxicity endpoint: Adequate

Study 1: Combined 28

- d ay repeated

- dose

, reproductive, and developmental oral toxicity study in rats (Product Safety Labs, 2012)

Title: Oxidized Castor Oil: Combined 28

- day Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor Oil, Oxidized ; CAS RN 68187

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- 8; Synonyms: Pale 16, Material

#104495

Remarks: Clear, yellow

- amber liquid with a mild odor, Batch #0000113649, puri ty
100%

METHOD

Method/guideline followed: OECD TG 422

, OPPTS 870.3650

, TSCA 40 CFR

799.9365

Test type: Combined repeated

- dose toxicity study with the reproduction/developmental toxicity screening test

GLP compliant?: Yes

Year study performed: 2011

- 2012

Test species: Crl:CD ® (SD) IGS rats ; age: 10 weeks old at study initiation ; supplier: Charles River Laboratories, Inc.

Number of animals/sex/dose: 15 females/dose ( 5 were in general toxicity group and 10

in reproduction toxicity group) and 10 males/dose ( all males were used for mating; 5 males from this group were used to assess reproduction toxicity by evaluation of reproductive organs ; 5 from this group were randomly assigned for general toxicity evaluations)

Route of administration: Oral

- gavage

Vehicle: O live oil with

~ 5% ethanolFinal HPV 2 Data Adequacy Review CAS RN 68187

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10 Exposure duration: Females in reproduction subgroups: 2 weeks premating, up to 2 weeks mating, gestation, and lactation up to day 4 ; Females in g eneral toxicity female subgroup s : 37 days ; Males: 36 days

Doses/concentrations tested: 0, 250, 500 or 1000 mg/kg

- bw /day

Statistical methods used: A probability value of p < 0.05 was considered to be statistically significant.

In

‐ Life and Organ Weight Data (P arental Generation ):

“ 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

... Where Bartlett’s test indicates homogeneous

v ariances, treated and control groups were compared using a one

‐ way analysis of variance (ANOVA). When one way analysis of variance was significant, a comparison of the treated groups to control by Dunnett’s test

... for multiple comparisons was performed. W here variances are considered significantly different by Bartlett’s test, groups were compared using a nonparametric method (Kruskal

‐ Wallis non

‐ parametric analysis of variance

... ). When non

‐ parametric analysis of variance was significant, comparison of trea ted groups to control was performed using Dunn’s test

... Motor activity data was further evaluated by two

‐ way repeated measures analysis. ” [Product Safety Labs 2012 , p. 27 ]

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

... Group means are to be analyzed by a one

‐ way variance analysis (ANOVA) fol lowed by Dunnett's Test

... Alternatively, continuous data (e.g., clinical chemistry, hematology, etc.) determined to be nonhomogeneous, may be analyzed using a Kruskal

‐ Wallis Analysis of Variance

... followed by a pairwise Mann

‐ Whitney U Test

... For the Bartlet t 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. ” [Product Safety Labs 2012, p. 27]

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 independe ntly on all test groups with no inferential comparisons being made with regards to homogeneity of variance, normality, and analysis of variance. ” [Product Safety Labs

2012 , p. 28 ]

Test method/conditions remarks: “A combined repeated oral dose gavage toxicity study with a reproduction/developmental toxicity screening test was conducted inFinal HPV 2 Data Adequacy Review CAS RN 68187

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11 Crl:CD ® (SD) IGS rats to provide information on male and female toxicity and reproductive performance, including gonadal function , mating behavior, conception, development of conceptus’ [sic] and parturition following administration of Oxidized Castor Oil for at least 28 days. ” [ Product Safety Labs 2012, p. 14 ]

Rats (10 virgin males and 15 virgin females/dose) , which had been acclim ated for 7 days to experimental housing conditions, were administered the test substance (vehicle = olive oil containing ~5% ethanol) daily via gavage (dose volume = 5 mL/kg) at dose levels of 0 (Group 1; vehicle control), 250 (Group 2), 500 (Group 3)

, or 1000 (Group 4) mg/kg

bw /day . Ten animals/sex/dose were used for mating purposes. Each dose group was divided into two subgroups: reproductive and general toxicity

. T he “ reproductive toxicity ” subgroup ( 5 males and 10 females/dose) was used to determine effects on reproducti ve organs , while the “ general toxicity ” subgroup (5 non

- mated females /dose and 5 males/dose that were randomly selected from the group of mated males ) was used

for general toxicity evaluations

.

“ Parental/reproduction males and females received t h e test substance while housed separately for a 14

- day pre

- mating period, followed by dosing through a 14

- day [or less]

co

- habitation period. Upon determination of pregnancy or following t h e 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. [Five ] [m] ales [ at each dose] were sacrificed following the gestation phase and were evaluated for fertility, including spermatogenic stage and epididymal effects. A general toxicity cohort of males [5/dose that were randomly selected from the group of mated males ] and females were concomitantly administered t h e test substance and evaluated for a Functional

Observational Batter [y] and M otor Activity [Test] (FOB/MA, Day 35/33), clinical pathology (Days 34, hematology, clinical chemistry, urinalysis; Days 36/37, coagulation) and histopathologically following scheduled sacrifice on Day 36 (males) and 37 (females). Reproductive females were evaluated for fertility and reproductive parameters, including uterine and litter indices and sacrificed on Lactation Day 4. ” [Product Safety Labs 2012, p. 14]

Animals were fasted for ~18 hours prior to collection of blood and urine. Hematological parameters included erythrocyte count, hematocrit, mean corpuscular hemoglobin, absolute reticulocyte count, total white blood cell and differential leukocyte count, hemoglobin concentration, mean corpuscular volume, red cell distribution width, platelet

c ount, mean corpuscular hemoglobin concentration , prothrombin time, and active partial thromboplastin time

. Clinical chemistry parameters included serum aspartate aminotransferase, sorbitol dehydrogenase, total bilirubin, blood creatinine, triglycerides,

s erum protein, globulin, inorganic phosphorus, potassium, creatine phosphokinase, uric acid, serum alanine aminotransferase, alkaline phosphatase, urea nitrogen, total cholesterol, fasting glucose, albumin, calcium, sodium, chloride, and lactate dehydrogena se. Urinalysis parameters included quality, color, clarity, volume, microscopic urine sediment examination, pH, glucose, specific gravity, protein, ketone, bilirubin, blood, and urobilinogen.Final HPV 2 Data Adequacy Review CAS RN 68187

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12 “ Animals, bot h reproductive and general toxicity, were observe d daily for clinical signs

and mortality. The animals were observed for viability, signs of gross toxicity, and

behavioral changes at least once daily during the study, and approximately weekly for a battery of detailed observations. Individual body weig hts and food consumption were recorded weekly as prescribed for bot h sets of animals. Body weights for all females

were recorded on Gestation Days (GD) 0, 7, 14, and 20, and on Lactation Days (LD) 0

and 4. Food consumption for all males and females was r ecorded to coincide wit h body weight collection [with the exception of during the mating period]

. ” [Product Safety Labs 2012, p. 14]

Additional e ndpoints monitored in the parental generation included body weight gain, food efficiency, estrous cycle, matin g behavior , and gestation duration . Parental rats from the general toxicity study were subjected to a full necropsy and all major organs and tissues were prepared for histopathological examination.

R eproductive organs (including prostate, seminal vesicle, and coagulating gland [combined]; testes [combined]; epididymides [combined]; non

- gravid uterus; cervix; fallopian tubes; and ovaries [combined]) of rats from the reproductive toxicity groups

were weighed and prepared for histopathological examination. The uterus was examined for the number and position of conceptuses, implantation scars, and corpora lutea. Reproductive calculations included mating and fertility indices, fecundity, implantation index, pre

- and post

- im plantation loss es, gestation index, live birth index, stillborn index, and pup viability index.

In general toxicity groups, the following tissues were weighed: adrenals (combined), brain, epididymides, heart, kidneys (combined), liver, ovaries with ovid ucts, testes (combined), spleen, thymus, and uterus with cervix. The following tissues and organs from general toxicity groups were prepared for histopathological examination: seminal vesicles (with coagulating glands), adrenals, all gross lesions, aorta, bone (femur), brain (three sections: cerebrum, medulla/pons, and cerebellum), peripheral nerve (sciatic), jejunum, kidneys, larynx, liver, lungs, mammary gland, nose and nasal turbinates, ovaries and oviducts, pancreas, trachea, skeletal muscle, skin, spi nal cord (three levels: cervical,

mid

- thoracic, and lumbar), spleen, sternum, stomach, thymus, thyroid/parathyroid, cecum, colon, duodenum, esophagus, Harderian gland, heart, ileum with peyer’s patches, pharynx, pituitary, rectum, lymph node mesenteric, ly mph node mandibular, bone marrow collected with femur and sternum, salivary glands (sublingual, submandibular, and parotid), urinary bladder, uterus with cervix, vagina, prostate, epididymides, eyes with optic nerve, and testes.

“ Histopathology examina tion 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 te sticular 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 aFinal HPV 2 Data Adequacy Review CAS RN 68187

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13 Group 2 female (7049), the uterus of each of two Group 3 f emales (7071 and 7072), and

the esophagus of a Group 3 male (7055). ” [P roduct Safety Labs 2012, p. 26]

Litter parameters (including litter size, number of stillborn pups, number of live born pups, sex ratio, and total number of pups per litter) were recorded immediately after birth. Pups were weighed and observed for clinical signs and external abnormalities. At

termination on LD 4

, pups were weighed, sexed, and sacrificed. T he thorax, abdomen, pelvis, and palate of a ll pu ps that were found dead or sacri fi ced were examined internally. Pups with abnormalities or gross lesions were preserved for verification of abnormalities.

“ The test substance was mixed weight to volume (w/v) in vehicle. The dose mixtures

were maintained on a magnetic stir plate during dose administration [and used within ~ 2 hours of preparation]

. Fresh dilutions

... of the test substance were prepared daily. The control group received vehicle only at the same volume as the test animals. As a

facilitatio n of the mixing process, a quantity of ethanol approximately 5% v/v of the total volume to be prepared was added to the test substance or control mixture (i.e., 2.5 mL of ethanol was added to the mixture if a 50 mL final volume was prepared). The test

sub stance and ethanol were mixed prior to adding the olive oil. ” [Product Safety Labs 2012, p. 20 ]

“A sample of the neat test substance was collected at the beginning of the study and again following the completion of the in

- life phase of the study and analyzed by Product Safety Labs to evaluate the stability of the test substance over the course of the study. Chemical analysis was also conducted on dose formulation solutions to verify dose concentrations (Days 1, 21, and 42) and homogeneity (Day 1). B ased on the stability and the concentration verification results, the animals are considered to have received the targeted concentrations of 50, 100, and 200 mg/mL of Oxidized Castor Oil. All dose preparations were considered to be homogeneously distribut ed [based on measurements at the top, middle, and bottom of dosing solutions] .” [Product Safety Labs 2012, p. 14]

“The test substance was considered stable under the conditions of storage over the course of this study. Based on the stability, homogeneit y and dose concentration verification results, the animals are considered to have received the targeted dose concentrations of 250, 500, and 1000 mg /kg/ day Oxidized Castor Oil.” [Product Safety Labs 2012, p. 15]

RESULTS

Endpoint value(s):

NOAEL (repeated

- dose toxicity) = 1000 mg/kg

- bw/day; highest dose tested. NOAEL ( reproductive toxicity) = 1000 mg/kg

- bw/day; highest dose tested. NOAEL ( developmental toxicity) = 1000 mg/kg

- bw/day; highest dose tested.

Results remarks: “ There were no test subst ance

- related or other mortalities of the parental animals during the course of the study. ” [Product Safety Labs 2012, p. 31]Final HPV 2 Data Adequacy Review CAS RN 68187

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14 N o clinical signs in males or females (reproductive and general toxicity groups) were considered to be attributable to the test substance at any stage throughout the course of the study. Any changes were considered to be incidental and toxicologically insignificant.

“ In general, the functional behavioral results of the general toxicity test groups of male and female rats performe d 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 toxicologica lly significant behavioral change. ” [ Product Safety Labs 2012, p. 3 2 ]

“ 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

f

e male test groups compared to their corresponding control groups. ” [Product Safety

Lab s 2012, p. 3 3 ]

There were no changes in body weight

, body weight gain , food consumption, or food efficiency in either sex that were considered to be attributable to the test substance

.

“ 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 .” [Product Safety Labs 2012, p. 3 5 ]

The following stat istically significant changes in clinical chemistry were o bserved, but

were considered to be toxicologically insignificant because they were not accompanied by any other corresponding clinical or histopathologic al change: slightly reduced potassium concentration in males at 250 mg/kg

- bw/day, slightly increased ur ea nitrogen

concentration in males at 250 , 500, and 1000 mg/kg

- bw/day, slightly decreased triglycerides in females at 500 and 1000 mg/kg

- bw/day, and slightly decreased uric acid concentration in females at 1000 mg/kg

- bw/day

.

The following statistically significant changes in hematology were observed, but were considered to be toxicologically insignificant because they were not accompanied by any other corresponding clinical or histopathological change: slightly increased hemog lobin distribution width in females at 250 mg/kg

- bw/day, slightly decreased eosinophil concentration in males at 500 mg/kg

- bw/day, slightly decreased basophil concentration in females at 250, 500, and 1000 mg/kg

- bw/day, slightly decreased absolute basophil concentration in females at 250 and 1000 mg/kg

- bw/day, and slightly decreased absolute segmented neutrophil concentration in females at 250 and 1000 mg/kg

- bw/day.

“ 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 consideredFinal HPV 2 Data Adequacy Review CAS RN 68187

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15 incidental and non

- adverse as they were not accompanied by any other clinical or histologic al change. As such, these and all other changes were considered toxicologically insignificant. ” [Product Safety Labs 2012, p p . 3 6

- 37 ]

Macroscopic Findings: “ At scheduled sacrifice [on] 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. ” [Product Safety Labs 2012, p. 37]

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, Oxidiz ed Castor

Oil ... There was no testicular toxicity related to administration of the test substance.” [Product Safety Labs 2012, p. 37]

Organ Weight and Organ Weight Ratios: “ All organ weights for the adult treated reproductive and general toxicity animal s 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

... 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. ” [Product Safety Labs 2012, p. 3 8 ]

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. ” [Product Safety Labs 2012, p. 38]

There w ere no treatment

- re lated effect s on estrus cycle, pre

- coital length

, gestational length, fertility, mating index , fecundity, gestation index, numbers of implantation sites and corpora lutea

, early and late resorptions, pre

- and post

- implantation loss , mean litter size and weight, sex ratio, pup viability, and stillbirth and live birth indices

.

Offspring Generation (F1)

Individual F1 Fate: “ There were no F1 clinical findings or congenital anomalies deemed

related to the administration of the test s ubstance, 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 incompatible 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. [This included:] Three of 132 (2.3%), 1/141 (0.7%), 4/135 (3.0%), and 3/131 (2.3%) in Groups 1

- 4, respectively. ” [Product Safety Labs 2012, p. 40 ]

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 4Final HPV 2 Data Adequacy Review CAS RN 68187

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16 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. ” [Product Safety Labs 2012, p p . 40

- 41 ]

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 , c orresponding 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 tre ated groups when compared to controls. ” [Product Safety Labs 2012, p. 4 1 ]

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. ” [Product Safety Labs 2012 , p. 41 ]

CONCLUSIONS

“ 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 o f the stages of spermatogenesis and interstitial testicular cell structure, revealed no test s ubstance

- related effects. Furthermore, the filial generation from all test groups exhibited normal parameters in accordance with that expected for this strain of rat

... 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 cond itions 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 re production/developmental screening.” [Product Safety Labs 2012 , p. 41 ]

STUDY RELIABILITY

[1] Reliable without restrictions.

REFERENCES

Product Safety Labs. 2012. Oxidized Castor Oil: Combined 28

- day Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats. Sponsor: Vertellus Specialties Inc., Indianapolis, IN.

Text in quotes was taken directly from the report of Pro duct Safety Labs (2012).Final HPV 2 Data Adequacy Review CAS RN 68187

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17 D

- 6 . Genetic Toxicity

– Gene Mutation

Summary of endpoint: Adequate

Study 1: Mutagenicity studies in Salmonella typhimurium and Escherichia coli (Bioservice Scientific Laboratories, 2011)

Title: Reverse Mutation Assay using Bacteria ( Salmonella typhimurium and Escherichia

coli ) with Oxidized Castor Oil (aka Pale 16, Material #104495)

This study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor Oil, Oxidized ; CAS RN 68187

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- 8; Synonyms: Pale 16, Material

#104495

Remarks : Yellow

- amber liquid; Batch No. 0000113649; purity: 100%

METHOD

Method/guideline followed: OECD TG 471, EC No. 440/2008 B.13/14, OPPTS 870.5100, TSCA 40 CFR 799.9510

Test type: Bacterial Reverse Mutation Test

GLP compliant?: Yes

Year study performed: 2011

Test species: Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537;

E

scherichia coli strain WP2 uvrA.

Metabolic activation: With and without

Concentrations tested: 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

Statistical methods used: None specified

Test method/conditions remarks : “In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate.” [Bioservice Scientific Laborator ies 2011, p. 12] One experiment used the plate incorporation method, while the other used the pre

- incubation method (test item preparation incubated for 60 minutes at 37 °C before adding to overlay agar).Final HPV 2 Data Adequacy Review CAS RN 68187

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18 “Tester strains TA 98, TA 100, TA 1535, TA 1537 and E. coli [were] obtained from MOLTOX, INC., NC 28607, USA. They [were] stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (approx. 8% v/v) over liquid nitrogen.” [Bioservice Scientific Laboratories 2011, p. 16]

“S amples of each tester strain were grown by culturing for 12 h at 38.5 °C in S. typhimurium medium (Nutrient Broth) and E. coli medium (Luria Bertani), respectively, to the late exponential or early stationary phase of growth (approx. 10 9 cells/mL).” [Bioservice Scientific Laboratories 2011, p. 16]

“The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β

- naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.” [Bioservice Scientific Laboratories 2011, p. 17]

“The test item was dissolved in DMSO and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and S9 activity.” [Bioservice Scientific

Laboratories 2011, p. 14]

Vogel

- Bonner Medium E agar plates with 2% glucose were used in the Ames test. After the overlay agar was poured on the surface, plates were inverted and incubated at 37 °C

for at least 48 hours in the dark.

“Negative and/or solvent as we ll as positive controls were included in each experiment. Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.” [ Bioservice Scientific Laboratories 2011, p. 14]

Positive controls without activation: TA100, TA1535: Sodium azide (10 μg/plate)
TA98, TA1537: 4

- Nitro

- o

- phenylene

- diamine (10 μg/plate for TA98, 40 μg/plate for TA1537) WP2 uvrA: Methylmethanesulfonate (1 μL/plate)

Postive control with activation:

All strains: 2

- Aminoanthracene (2.5 μg/plate for TA98, TA100, TA1535, and TA1537; 10 μg/plate for WP2 uvrA)

Cytotoxicity was determined by a “clearing or rather diminution of the background

lawn... or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0 .5 in relation to the solvent control.” [ Bioservice Scientific Laboratories 2011, p. 20]

The test was considered valid if: (1) TA98 and TA100 demonstrated the appropriate response to ampicillin; (2) the control plates with and without S9 exhibited sponta neous reversion frequencies within the historical control range; (3) corresponding background growth on both negative control and test plates was observed; and (4) positive controls

exhibited a distinct enhancement of revertant rates over the controls.Final HPV 2 Data Adequacy Review CAS RN 68187

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19 Th e test item was considered mutagenic if “a clear and dose

- related increase” in the number of revertants occurred and/or a “biologically relevant positive response” occurred for at least one of the dose groups in at least one tester strain with or without a ctivation.
[ Bioservice Scientific Laboratories 2011, p. 20] A biologically relevant increase was defined as an increase in the number of revertants of at least 2x in TA98, TA100, and WP2 uvrA or at least 3x in TA1535 and TA1537, as compared to the solvent control.

RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable): “No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation.” [Bioservice Scientific Laboratories 2011, p. 12]

Results remarks: “No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with O xidized Castor Oil (aka Pale 16, Material # 104495) at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.” [Bioservice Scientific Laboratories 2011, p. 12]

“The reference mutagens induced a distinc t increase of revertant colonies indicating the validity of the experiments.” [ Bioservice Scientific Laboratories 2011, p. 12]

“ No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic act ivation).” [Bioservice Scientific Laboratories 2011, p. 12]

CONCLUSIONS

“[I]t can be stated that during the described mutagenicity test and under the experimental conditions reported, Oxidized Castor Oil (aka Pale 16, Material #104495) did not cause

gene mutation by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Oxidized Castor Oil (aka Pale 16, Material #104495) is considered to be

non

- mutagenic in this bacterial reverse mutation assay.” [ Bioservice Scientific

Labo ratories 2011, p. 12]

STUDY RELIABILITY

[1] Reliable without restrictions.Final HPV 2 Data Adequacy Review CAS RN 68187

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20 REFERENCES

Bioservice Scientific Laboratories. 2011. Reverse Mutation Assay using Bacteria ( Salmonella typhimurium and Escherichia coli ) with Oxidized Castor Oil (aka Pale 16, Material #104495). BSL study No. 114188. Sponsor: Vertellus Specialties Inc., Indianapolis, IN.

Text in quotes was taken directly from the report of Bioservice Scientific Laboratories ( 2011 ).Final HPV 2 Data Adequacy Review CAS RN 68187

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21 D

- 7 . Genetic Toxi city

– Chromosomal Aberrations

Summary of endpoint: Adequate

Study 1: Chromosomal aberrations study in Chinese Hamster V79 cells (Bioservice Scientific Laboratory, 2012)

Title: In vitro Mammalian Chromosome Aberration Test in Chinese Hamster V79 cells with Oxidized Castor Oil (aka Pale 16, Material #104495)

The study is: Adequate Not Adequate

TEST SUBSTANCE

Identity: Castor Oil, Oxidized ; CASRN 68187

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- 8; synonyms: Pale 16, Material

#104495

Remarks: Yellow

- amber liquid, Batch No. 0000113649; purity: 100%

METHOD

Method/ guideline followed: OECD TG 473 , EC No. 440/2008 B.10, OPPTS 870.5375,

TSCA 40 CFR 799.9537

Test type: In vitro mammalian chromosomal aberration test

GLP compliant ?: Yes

Year study performed: 2011

Test species: Chinese Hamster V79 cells

Metabolic activation : With and without

Concentrations tested: Exposure concentrations : Experiment I: 15.6, 31.3, 62.5, 125, 200, 250, 350, and 500 μg/mL (without metabolic activation); 62.5, 125, 250, 500, 1000, 2500, and 5000 μg/mL (with metabolic activation). Experiment II: 37.5, 75, 150, 200, 225, 250, 300, 350, 400, 500, and 600 μg/mL (without metabolic activation); 200, 400, 800, 1200, 2000, 3000, 4000, and 5000 μg/mL (with metabolic activation). Concentrations selected for analysis of chromosomal aberra tions : Experiment I: 62.5, 200, 250, 350, and 500 μg/mL (without metabolic activation); 62.5, 500, 2500, and 5000 μg/mL (with metabolic activation).Final HPV 2 Data Adequacy Review CAS RN 68187

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22 Experiment II: 225, 350, and 500 μg/mL (without metabolic activation); 200, 1200, 4000, and 5000 μg/mL (with metabolic activation).

Statistical methods used: None specified

Test method/conditions remarks: In two experiments, Chinese hamster V79 cells were exposed to the test item. “The chromosomes were prepared 20 h after start of treatment

with the te st item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and 20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100

metap hases per culture were scored for structural chromosomal aberrations.” [ Bioservice Scientific Laboratories 2012, p. 13] The concentrations were selected for microscopic analysis of chromosomal aberrations based on mitotic index.

The stock cultures of V79 (ATCC, CCL

- 93) cells were stored over liquid nitrogen in the cell bank of BSL BIOSERVICE. For the experiments, thawed cells were seeded in minimum essential medium (MEM) supplemented with fetal bovine serum (FBS) at 37 °C in a 5% carbon dioxide atmospher e (95% air) and subcultures were made every 3

- 4 days.

“The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β

- naphthoflavone (100 mg/kg bw) for three consecutive days b y oral route.” [ Bioservice Scientific Laboratories 2012, p. 18]

“The test item was dissolved in DMSO followed by ultrasound for around 3

- 4 minutes.

To reach a final concentration of 1% solvent v/v in the treatment medium the solution

was diluted in ce ll culture medium (MEM) and treated with ultrasound for [an] additional few minutes. The solvent was compatible with the survival of the cells and the S9 activity.” [ Bioservice Scientific Laboratories 2012, p. 16]

“Positive and negative controls were included in each experiment... A negative control, consisting of cell culture medium (solvent or vehicle) alone and treated in the same way as the treatment groups was included.” [ Bioservice Scientific Laboratories, 2012

, p . 16 ] Ethylmethanesulfonate (EMS; 400 and 600 μg/mL) and cyclophosphamide (CPA; 0.83 μg/mL) were used as positive controls without and with metabolic activation, respectively.

Prior to harvesting, cell cultures were treated with Colcemid ® for 2.5 hours. Cells were examined microscopically and “structural chromosomal aberrations, including breaks, fragments, deletions, exchanges and chromosomal disintegration were recorded. Gaps

were recorded as well but not included in the calculation of the aberration rates... At least 200 well spread metaphases per concentration and validity controls were scored for cytogenetic damage.” [ Bioservice Scientific Laboratories 2012, p. 20]Final HPV 2 Data Adequacy Review CAS RN 68187

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23 “To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined by counting the number of mitotic cells in 1000 cells. Additionally the number of polyploid cells was scored... As an additional parameter for cytotoxic effects of the test item, the relative cell density was calculated as the mean of twent y cell counts per test group (cells

within the visual field at a 400

- fold magnification).” [ Bioservice Scientific Laboratories 2012, pp. 20

- 21]

The assay was considered acceptable if “the number of aberrations found in the negative

and/or solvent controls falls within the range of historical laboratory control data: 0.0%

- 4.0% (with and without activation) [and] the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome

aberrations.” [ Bi oservice Scientific Laboratories 2012, p. 21]

The criteria for determining a positive result were “a clear and dose

- related increase in the number of cells with aberrations [and] a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0%

- 4.0% aberrant cells (with and without activation)).” [ Bioservice Scientific Laboratories 2012, p. 21] The result was considered to be negative if there was “no biologically

relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group”. [ Bioservice Scientific Laboratories 2012, p. 21]

RESULTS

Genotoxicity result: Negative with and without metabolic activation

Cytotoxic concentration (if applicable) : ≥ 350 μg/mL without activation; 5000 μg/mL

with activation

Results remarks: “In both experiments no biologically relevant increase of the

aberration rates was noted after treatment with the test item with and without metabolic

activation. The aberra tion rates of all dose groups treated with the test item were within the historical control data of the negative control. In the experiments I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid c ells was found after treatment with the test item as compared to the controls.” [Bioservice Scientific Laboratories 2012, p. 13]

“In experiment I precipitation of the test item was noted without metabolic activation at concentrations of 125 μg/mL and higher, with metabolic activation at concentrations of 250 μg/mL and higher. In experiment II precipitation of the test item was noted without

metabolic activation at all concentrations evaluated , with metabolic activation at concentrations of 400 μg/mL and higher.” [Bioservice Scientific Laboratories 2012, p. 13]

The positive controls “induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.” [Bioservice Scientific Laboratories 2012, p. 13]Final HPV 2 Data Adequacy Review CAS RN 68187

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24 CONCLUSIONS

“[ I]t can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Oxidized Castor Oil (aka Pale 16, Material #104495) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item Oxidized Castor Oil (aka Pale 16, Material #104495) is considered to be non

- clastogenic in this chromosome aberration

test.” [ Bioservice Scientific Laboratories 2012, p. 14]

STUDY RELIABILITY

[1] Relia ble without restrictions.

REFERENCE

Bioservice Scientific Laboratories. 201 2

. In vitro Mammalian Chromosome Aberration

Test in Chinese Hamster V79 cells with Oxidized Castor Oil (aka Pale 16, Material #104495). BSL study No. 11418 9 . Sponsor: Vertellus Specialties Inc., Indianapolis, IN.

Text in quotes was taken directly from the report of Bioservice Scientific Laboratories ( 2012 ).Final HPV 2 Data Adequacy Review CAS RN 68187

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25 E. DATA ADEQUACY SUMMARY TABLE

Endpoint Study Information

Available Acceptable Data Gap

Aquatic Effects Acute Toxicity to Fish No

– No 1

Acute Toxicity to Aquatic

Invertebrates No

– No 1

Toxicity to Aquatic Plants No

– No 1

Health Effects Acute Toxicity Yes Yes No Repeated

- Dose Toxicity Yes Yes No

Gene Mutation Yes Yes No

Chromosomal Aberrations Yes Yes No

Reproductive Toxicity Yes Yes No Developmental Toxicity Yes Yes No

1 EPA is not requiring aquatic toxicity testing of CASRN 68187

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- 8; this testing is not considered feasible due to the extremely poor water solubility of this substance.