butanedioic acid, 2-octenyl

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: USEPA OPPTS 870.3650 (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Justification for study design:

Route, Method of Administration, Frequency, Duration and Justification:
Oral gavage was the preferred route of exposure according to the relevant test guideline. Male rats were dosed daily for 14 days prior to mating and continuing throughout mating for 35 days. Female rats were dosed once daily for 14 days prior to breeding, and continuing through breeding (two weeks), gestation (three weeks), and lactation (four days).

Dose Levels and Justification:
Selection of the high-dose level of 100 mg/kg body weight/day was based upon the stomach gross and histopathological data obtained from the range-finding study (Hannas et al., 2016) and was expected to induce some toxic effects, but not death or obvious suffering. The lower dose levels were expected to provide dose response data for any toxicity observed among the high-dose animals, and to establish a no-observed-effect level (NOEL).

Test material

Test material information

Constituent 1

Reference substance name:

butanedioic acid, 2-octenyl

EC Number:

820-780-3

Cas Number:

62568-82-5

Molecular formula:

C12 H20 O4

IUPAC Name:

butanedioic acid, 2-octenyl

Test material form:

other: White solid

Details on test material:

Test Item Name : Octenylsuccinic Acid
Appearance/Colour/Odour : White solid
Lot N° : 8515025

Specific details on test material used for the study:

Test Material Name: Octenylsuccinic acid
Chemical Name: 2-(Octen-1-yl) butanedioic acid
Synonyms: DF-20 Acid, OSAC
Lot/Reference/Batch Number: 8515025
Purity/Characterization (Method of Analysis and Reference): The purity of the test material was determined to be 99.2% area by high performance liquid chromatography with identification by liquid chromatography mass spectrometry
and nuclear magnetic resonance (Megregian, 2016).
Test Material Stability Under Storage Conditions: Octenylsuccinic acid, lot 8515025, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under U.S. EPA OPPTS Guideline 830.6313 (Megregian and Crispin, 2016).

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and Sex: Rats (male and female)
Strain and Justification: Crl:CD(SD) rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data and the reliability of the commercial supplier.
Supplier and Location: Charles River (Raleigh, North Carolina)
Age at Study Start: Approximately eight weeks of age at initiation of treatment.

Physical and Acclimation:
During the acclimation period each animal was evaluated by a veterinarian trained in the field of Laboratory Animal Medicine, or a trained animal/toxicology technician, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory was fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals were housed two-three per cage in stainless steel solid bottom cages with corncob bedding, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), prior to randomization. Animals were acclimated to the laboratory for at least one week prior to the start of the study.

Housing:
After assignment to study, animals were housed one per cage in stainless steel cages, except during breeding and during the gestation and littering phases of the study. Cages had solid floors with corncob bedding. During breeding, one male and one female were placed in stainless steel cages with wire mesh floors that were suspended above absorbent paper in order to better visualize copulation and plugs. After breeding, males were returned to solid bottom stainless steel cages. During gestation and littering, dams (and their litters) were housed in plastic cages provided with irradiated ground corn cob nesting material from approximately GD 0 until LD 4. Cages contained a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions were maintained in the animal room.
Temperature: 22°C with a range of 20°C-26°C
Humidity: 50% with a range of 30-70%
Air Changes: 10-15 times/hour (average)
Photoperiod: 12-hour light/dark (on at 6:00 a.m. and off at 6:00 p.m.)
Temporary excursions from these ranges for temperature and humidity may have occurred on an infrequent basis; all observed ranges were documented in the study file.

Randomization and Identification:
Prior to test material administration, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alphanumeric identification numbers.

Feed and Water:
Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water were provided ad libitum. Analyses of the feed were performed by PMI Nutrition International to confirm the diet provided adequate nutrition and to quantify the levels of selected contaminants. Drinking water obtained from the municipal water source was periodically analyzed for chemical parameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants were conducted at periodic intervals by an independent testing facility. There were no contaminants in either the feed or water at levels that would have adversely impacted the results or interpretation of this study. Copies of these analyses were maintained in the study file.

Animal Welfare:
In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities were in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activity to be used for this study was DART 01.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Dose Preparation:
The test material was administered in a propylene glycol (PG) vehicle, such that a dose volume of 6 ml/kg body weight yielded the targeted dose. Dose volumes were adjusted using the most current body weight. Dose suspensions were prepared periodically throughout the study period based upon stability.

Solubility:
Octenylsuccinic acid was determined to be soluble in propylene glycol at a concentration of 250 mg/ml.

Details on mating procedure:

Breeding Procedure:
Breeding of the adults commenced after approximately two weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until pregnancy occurred or two weeks had elapsed. During the breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then separated from the males and returned to their home cages. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis:
Dose Confirmation and Homogeneity:
Analysis to determine concentration of the test material of all dosing suspensions from the first mix of the main study was initiated prior to the start of dosing. The
low- and high-dose suspensions from the first mix of the main study were analyzed to confirm homogenous distribution of the test material concurrent with dose
confirmation. Analyses were conducted using High Performance Liquid Chromatography/Mass Spectrometry (HPLC/MS).

Stability:
Stability of the test material in the vehicle was determined prior to the start of the study at concentrations ranging from 2.5-250 mg/ml (Marty and Hales, 2017). The established concentration range and duration spanned those used in this study.

Retainer Samples:
A sample of the solid test material was retained, but samples of the dose suspensions were not retained.

Duration of treatment / exposure:

Groups of 12 male and 12 female Crl:CD(SD) rats were administered octenylsuccinic acid in propylene glycol daily, by gavage, at dose levels of 0 (control), 10, 30, or 100 mg/kg/day. Female rats were dosed once daily for approximately two weeks prior to breeding, continuing through breeding (two weeks), gestation (three weeks), and through postpartum day 4. Male rats were dosed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 36).

Frequency of treatment:

Rats were administered octenylsuccinic acid in propylene glycol daily, by gavage.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12/sex/dose group

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Daily Observations:
A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. The animals were not hand-held for these observations unless deemed necessary. Significant abnormalities that could have been observed included, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Cage-side examinations were conducted on dams and their litters, at least twice daily. These examinations were conducted as described above.

Clinical Observations:
Clinical observations were conducted on all animals pre-exposure and at least daily throughout the study. During the exposure period, these examinations were conducted approximately one hour after dosing. Females were observed for signs of parturition beginning on or about gestation day (GD) 20 (see litter data). Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

Detailed Clinical Observations:
Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study. Mated (sperm-positive or plug-positive) females received DCO examinations on GD 0, 7, 14, and 20. Females that delivered litters were subsequently evaluated on LD 3. Detailed clinical observations were not conducted on females that failed to mate or deliver a litter during the gestation and lactation phases of the study. The DCO was conducted at approximately the same time each examination day, according to an established format. The examination included cage-side, hand-held and open-field observations, which were recorded categorically or using explicitly defined scales (ranks).

Functional Tests:
The functional tests (sensory evaluation, rectal temperature, grip performance and motor activity) were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on LD 4. Females that failed to deliver did not undergo functional testing during the last week of treatment.

Body Weights/Body Weight Gains:
All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Male body weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on GD 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate or deliver a litter were weighed at least weekly for the remainder of the study. Body weight gains were determined during the pre-mating phase and for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

Feed Consumption:
Feed consumed was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and end of a measurement cycle. During breeding, feed consumption was not measured for males or females due to cohousing. Following breeding, feed consumption was not measured for males. For mated females, feed consumption was measured on GD 0, 7, 11, 14, and 20. For females delivering litters, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock)/(# of days in measurement cycle)

Clinical Pathology:
Animals were fasted overnight prior to blood collection. Blood samples were obtained from the orbital sinus following anesthesia with a mixture of isoflurane vapors and medical oxygen at the scheduled necropsy. Blood was not obtained from animals that died or were euthanized in a moribund condition prior to their scheduled necropsy. Blood samples were not obtained from females that failed to deliver a litter. Hematology, coagulation and clinical chemistry parameters were evaluated.

Urinalysis:
Urine samples were obtained from all males the week prior to the scheduled necropsy. Animals were housed in metabolism cages and the urine collected overnight (approximately 16 hours). Feed and water were available during this
procedure.

Microscopic examination of urine:
Urine samples were collected from each male by manual compression of the urinary bladder. The urine samples were pooled from each group, and the microsediments were characterized microscopically.

Litter observations:

Litter Data:
Females were observed for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day the presence of the litter was noted and was designated as LD 0. All litters were examined as soon as possible after delivery. The following information was recorded on each litter: date of parturition, litter size on the day of parturition (LD 0), the number of live and dead pups on days 0, 1, and 4 postpartum, and the sex and the weight of each pup on LD 1 and 4. Any visible physical abnormalities or demeanor changes in the neonates were recorded as they were observed during the lactation period (see Daily Observations). In addition, litter observations were recorded on LD 0, 1, and 4. Any pups found dead were sexed and examined grossly, if possible, for external and visual defects and then discarded.

Postmortem examinations (parental animals):

Anatomic Pathology:
Adult Necropsy:
Adult males (fasted) were submitted for necropsy after at least four weeks of exposure. Adult females (fasted) were terminated on lactation day 5 or at least 24 days after the end of the mating period for females not producing a litter. On the morning of the scheduled necropsy, rats were weighed in the animal room and submitted alive for necropsy. The animals were anesthetized with a mixture of isoflurane vapors and medical grade oxygen. While under anesthesia, blood was collected from the orbital sinus (all males, all females that littered). The animals were placed in a CO2 chamber to continue anesthesia. Under a deep plane of anesthesia, their tracheas were exposed and clamped, and the animals were euthanized by decapitation.
A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain based on Kopf et al. (1964) and examined for the presence and number of implantation sites. Uteri were gently rinsed with saline and preserved in neutral phosphate-buffered 10% formalin.
Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, and thyroids with parathyroids (weighed after fixation) were recorded, and organ: body weight ratios calculated.
Representative samples of tissues listed in Table 1 were collected and preserved in neutral, phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s fixative. Transponders were removed and placed in jars with the tissues.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for these animals except that terminal body and organ weights were not recorded.

Histopathology:
Histologic examination of the tissues, indicated in the OECD 422 Attachment (Table 4), was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose (kidney and stomach) and relevant gross lesions. Paraffin embedded tissues were sectioned approximately 6 μm thick, stained with hematoxylin and eosin and examined by a veterinary pathologist using a light microscope.
The histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes were examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).
Selected histopathologic findings were graded to reflect the severity of specific lesions to evaluate: 1) the contribution of a specific lesion to the health status of an animal, 2) exacerbation of common naturally occurring lesions as a result of the test material, and 3) dose-response relationships for treatment-related effects. Very slight and slight grades were used for conditions that were altered from the normal textbook appearance of an organ/tissue, but were of minimal severity and usually with less than 25% involvement of the parenchyma. This type of change was neither expected to significantly affect the function of the specific organ/tissue nor have a significant effect on the overall health of the animal. A moderate grade was used for conditions that were of sufficient severity and/or extent (up to 50% of the parenchyma) that the function of the organ/tissue was adversely affected, but not to the point of organ failure. The health status of the animal may or may not have been affected, depending on the organ/tissue involved, but generally lesions graded as moderate were not life threatening. A severe grade was used for conditions that were extensive enough to cause significant organ/tissue dysfunction or failure. This degree of change in a critical organ/tissue was life threatening.
A complete set of tissues (Table 4) was examined from rats found dead or moribund. Histological examination was conducted in a similar manner as described above.

Postmortem examinations (offspring):

Anatomic Pathology:
Off-Spring Necropsy:
All pups surviving to lactation day 4 were euthanized by an oral dose of sodium pentobarbital solution, examined for gross external alterations, and then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.

Statistics:

The following statistical analysis were employed:
Descriptive statistics, Bartlett's test, parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA), Dunnett's test, Wilcoxon Rank-Sum, Bonferroni's correction, nonparametric ANOVA, sequential method of Grubbs, Fisher exact probability test, binomial distribution test, censored Wilcoxon test, z-test, analysis of covariance (ANCOVA), Pillai Trace statistic and repeated-measure design. A more detailed description of the statistics can be found in the "Other information on materials and methods" section.

Reproductive indices:

Reproductive indices were calculated for all dose level groups as follows:
• Female mating index = (No. females with evidence of mating/No. paired) x 100
• Male mating index = (No. males mated/No. paired) x 100
• Female conception index = (No. females with evidence of pregnancy/No. mated) x 100
• Male conception index = (No. males siring a litter/No. mated) x 100
• Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100
• Male fertility index = (No. males siring a litter/No. paired) x 100
• Gestation index = (No. females delivering a litter/No. females with evidence of pregnancy) x 100
• Gestation survival index = percentage of delivered pups alive at birth
• Post-implantation loss = (No. implants – No. offspring)/(No. implants) x 100
• Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

In-Life Observations:
No treatment-related effects on behavior or demeanor were observed at any dose level during the cage-side observations.

Detailed Clinical Observations:
Examinations performed on all animals prior to the study revealed that all animals were in good health for study purposes. Examinations performed on all animals weekly throughout the study revealed no treatment-related findings.

Mortality:

no mortality observed

Description (incidence):

One female given 100 mg/kg/day was euthanized on test day 22, due to inadvertent gavage complications (pulmonary edema, and inflammation of the trachea, bronchi and lung). All other animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No significant differences in body weights were observed for males at any dose level. Similarly, there were no treatment-related differences in the body weights or body weight gains of females at any dose level tested during the pre-mating, gestation, or lactation periods.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were no significant differences in the amount of feed consumed by females in any of the dose groups when compared to their respective controls throughout the study. Feed consumption was statistically identified as decreased for TD 1-4 (11.7%) and 4-8 (8.8%) in males given 30 mg/kg/day octenylsuccinic acid; however, this was deemed to be unrelated to treatment, as it was not dose-responsive. There were no significant differences in the amount of feed consumed by males in any of the dose groups when compared to their respective controls for the remainder of the prebreeding phase.

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology:
There were no statistically identified or treatment related hematologic effects in males or females at any dose level.

Prothrombin Time:
There were no statistically identified or treatment-related changes for males and females at any dose level.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Mean serum cholesterol for females given 100 mg/kg/day was statistically higher (34%) compared to controls and was interpreted to be treatment-related. The
slightly higher serum cholesterol level was interpreted to be non-adverse, because it was within the laboratory’s historical control range and moreover, there were no treatment-related histopathological changes in the liver.

Mean serum potassium concentrations were marginally higher (4%) and statistically identified in males in the 10 mg/kg/day and 100 mg/kg/day treatment groups as compared to the controls. This difference was interpreted to be unrelated to treatment due to lack of a dose-response relationship.

There were no other statistically identified or treatment-related serum clinical chemistry changes in males and females at any dose level.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

There were no statistically identified or treatment-related changes in urine volumes or specific gravity for males at any dose level.

The only treatment-related change was the decreased incidence of urinary pH 8.5 in males in the 30 mg/kg/day and 100 mg/kg/day treatment groups. While 10/12
control males had a urinary pH of 8.5, only 6/12 given 30 mg/kg/day and 1/12 given 100 mg/kg/day octenylsuccinic acid had a pH 8.5. This change was due to an
increasing trend toward pH of 8 or 7.5 in the 30 mg/kg/day group and pH 8.0, 7.5 or 7.0 in the 100 mg/kg/day group. The decreased incidence of urinary pH 8.5 in
males in the 30 mg/kg/day and 100 mg/kg/day treatment groups was interpreted to be due to possible urinary excretion of the test material and/or its metabolites, and hence, was considered a non-adverse effect.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional Tests:
Sensory Evaluation:
Examinations performed on males and females revealed no treatment-related findings in the sensory evaluation. There were two observations in females given 10 mg/kg/day that were statistically identified when compared to control (response to sharp noise, minimal; and response to sharp noise, moderate). These observations were considered spurious and unrelated to treatment due to the lack of a dose response relationship.

Rectal Temperature:
There were no treatment-related effects on rectal temperature either in males (p = 0.1371) or females (p = 0.9421).

Grip Performance:
There were no treatment-related effects on hindlimb grip performance either in males (p = 0.2548) or females (p = 0.5654). Similarly, there were no treatment-related effects on forelimb grip performance either in males (p = 0.5299) or females (p = 0.6038).

Motor Activity:
There were no treatment-related effects on motor activity. Treatment did not affect motor activity total counts (treatment x time interaction) either in males (p = 0.4491) or in females (p = 0.7992). The distribution of the motor activity counts within session (treatment x time x interval interaction) was not affected by treatment either in males (p = 0.2819) or in females (p = 0.3673).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related histopathological changes were noted in the stomachs of males in the 30 mg/kg/day and males and females in the 100 mg/kg/day treatment groups and the kidneys of males and females in the 100 mg/kg/day treatment group.

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive Indices, Pup Survival, and Sex Ratio:
There were no treatment-related effects at any dose level on any of the reproductive parameters or pup survival indices evaluated.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity as well as localized portal of entry (stomach) effects.

2

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity and portal of entry effects.

3

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

other: Neurological effects.

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no treatment-related effects on litter size or pup body weights at any dose level tested.

Effect levels (F1)

Remarks on result:

other: LOAEL/NOAEL were not determined for the offspring.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Any other information on results incl. tables

Analytical Chemistry:

Analyses of all dosing suspensions from the initial mix revealed mean concentrations ranging from 101 to 107% of targeted concentrations. Analyses of aliquots for the lowand high-dose suspensions indicated that the test material was homogeneously distributed based on relative standard deviations of ≤ 12.0%.

Histopathology Details:

Treatment-related histopathological changes were noted in the stomachs of males in the 30 mg/kg/day and males and females in the 100 mg/kg/day treatment groups and the kidneys of

males and females in the 100 mg/kg/day treatment group. These changes are summarized in Text Tables 6 and 7 for the stomach and kidneys, respectively.

Treatment-related stomach changes consistent with localized irritant effects of the test material were noted in males in the 30 mg/kg/day and 100 mg/kg/day treatment groups and in females in the 100 mg/kg/day treatment group. These effects were noted in, a) the limiting ridge of the forestomach (mucosal fold lined by keratinized stratified squamous epithelium representing the border between the non-glandular and glandular stomach), b) variably extending to other regions of the non-glandular mucosa, and c) the glandular stomach. A very slight hyperplasia of the limiting ridge epithelium was noted in males in the 30 mg/kg/day and 100 mg/kg/day treatment groups and females in the 100 mg/kg/day treatment group. As compared to the controls, this was a minimal change, characterized by a very slightly thickened stratified squamous epithelial cell layer thrown into multiple and slightly longer undulations of the basement membrane, variably forming rete-ridge like structures with or without slightly increased numbers of mitotic figures of the basal cell layer. The limiting ridge hyperplasia was accompanied by a very slight

hyperkeratosis characterized by a minimal increase in the amount of keratin on the surface of the limiting ridge epithelium in majority of the males given 100 mg/kg/day, 1/12 females given 100 mg/kg/day and in 1/12 males given 30 mg/kg/day. In addition, there was variable presence of multiple, round to ovoid spaces within the keratin layer (intracorneal bullae) filled with homogeneous eosinophilic material in some of these rats. Males given 100 mg/kg/day also had very slight multifocal vacuolization of the limiting ridge epithelium characterized by the presence of multifocal, clear, vacuolated squamous epithelial cells. Less frequently, squamous epithelial hyperplasia and hyperkeratosis extended to other regions of the non-glandular mucosa. One male given 100 mg/kg/day had slight, focally extensive, squamous epithelial hyperplasia and hyperkeratosis of the nonglandular mucosa. A very slight hyperkeratosis of the non-glandular mucosa was also noted in one female given 100 mg/kg/day. Other treatment-related changes within the non-glandular stomach included slight focally extensive edema and subacute to chronic inflammation of the non-glandular submucosa in two males given 100 mg/kg/day. Treatment-related microscopic changes were also noted in the glandular stomach which consisted of very slight, neck mucous cell hyperplasia of the glandular mucosa, particularly adjacent to the limiting ridge in males and females given 100 mg/kg/day and males given 30 mg/kg/day; and a very slight subacute to chronic inflammation in males and females given 100 mg/kg/day characterized by the presence of increased numbers of mononuclear cells and eosinophilic granulocytes in the glandular submucosa. All these histopathological changes noted in the stomach were interpreted to be localized responses to a repeatedly administered irritant test material at the portal of entry. There were no treatment-related histopathological effects in the stomach of males given 10 mg/kg/day or females given 10 mg/kg/day or 30 mg/kg/day.

In the kidneys, treatment-related changes were observed in a few rats given 100 mg/kg/day which were unilateral or bilateral in distribution. Female rats (5/12) and male rats (1/12) had unilateral or bilateral, very slight, multifocal hyperplasia and hypertrophy of the medullary tubular epithelium. This change was characterized by the presence of a few, multifocal basophilic tubules in the inner stripe of the outer medulla and occasionally within the inner medulla. These tubules were lined by slightly increased numbers of enlarged (hypertrophy) basophilic cells with slight nuclear crowding (hyperplasia), generally in a single layer with variable presence of mitotic figures. There were no associated degenerative or necrotic changes in these kidneys. Although the exact reason for these effects is not clear, they were likely related to possible acid-base regulation efforts by the kidney associated with the urinary excretion of the test material and/or its metabolites. There were no treatment-related histopathological changes in the kidneys of males and females given 10 mg/kg/day or 30 mg/kg/day.

One female rat given 100 mg/kg/day was euthanized on test day 22 due to inadvertent gavage complications. Following necropsy and histopathological evaluation of this animal, the cause of moribundity was attributed to pulmonary edema, inflammation of the trachea, bronchi and lung due to inadvertent complications of the gavage procedure, unrelated to the systemic toxicity of the test material.

All other histopathological findings were interpreted to be spontaneous background alterations unassociated with the exposure to octenylsuccinic acid.

The following tables have been included in the OECD 422 Attachment:

TABLE 4. Tissues Collected and Preserved at Necropsy

Text Table 5. Gross Pathologic Stomach Effect

Text Table 6: Treatment-related Histopathological Changes - Stomach

Text Table 7: Treatment-related Histopathological Changes - Kidneys

Applicant's summary and conclusion

Conclusions:

Based on these results, the lowest-observed-adverse-effect level (LOAEL) for systemic toxicity as well as localized portal of entry (stomach) effects was determined to be 100 mg/kg/day in males and females. The no-observed-adverse-effect level (NOAEL) for systemic toxicity and portal of entry effects was 30 mg/kg/day. The NOAEL for reproductive and neurological effects in males and females was 100 mg/kg/day, the highest dose level tested.

Executive summary:

The purpose of this study was to evaluate the potential effects of octenylsuccinic acid in rats following oral gavage administration on general toxicity, neurological and reproductive function, and prenatal/early neonatal growth and offspring survival. This study evaluated octenylsuccinic acid in the OECD 422 design.

Groups of 12 male and 12 female Crl:CD(SD) rats were administered octenylsuccinic acid in propylene glycol daily, by gavage at dose levels of 0 (control), 10, 30, or 100 mg/kg/day. Females were dosed once daily for two weeks prior to breeding, through breeding (up to two weeks), gestation (three weeks), and through postpartum day 4. Females were necropsied on postpartum day 5. Males were dosed for two weeks prior to breeding and continuing through breeding (two weeks) until necropsy (test day 36). Effects on general systemic toxicity, neurobehavioral assessments, clinical chemistry, hematology, coagulation, urine parameters, gonadal function, mating behavior, conception, development of the conceptus, parturition and early postnatal growth and survival were evaluated. In addition, a gross necropsy of the adults was conducted with extensive histopathologic examination of tissues. Litter size, pup survival, sex, body weight and the presence of gross external morphological alterations were assessed in the offspring.

Male and female rats administered 10, 30, or 100 mg/kg/day octenylsuccinic acid had no treatment-related effects in clinical signs, functional tests, motor activity, body weights/body weight gains, feed consumption, hematology, prothrombin time, and selected organ weights, as compared to control animals. There were no treatment-related effects of octenylsuccinic acid on reproductive function, or prenatal/early neonatal growth and survival of the offspring.

Female rats given 100 mg/kg/day had treatment-related, statistically higher (34%) mean serum cholesterol levels compared to controls. Urinalysis data indicated a dose-related decrease in the incidence of urine pH 8.5 in males given 30 or 100 mg/kg/day as compared to the controls and was interpreted to be due to possible urinary excretion of the test material and/or its metabolites, and hence, considered a non-adverse change.

Treatment-related gross pathological changes were confined to the stomach, the portal of entry. Males given 30 mg/kg/day and males and females given 100 mg/kg/day treatment groups had treatment-related, dose-dependent incidences of thickened limiting ridge of the forestomach. Multifocally thickened non-glandular mucosa of the forestomach was also observed in a few rats given 100 mg/kg/day.

Treatment-related histopathological effects were noted in the stomachs of males given 30 or 100 mg/kg/day and females given 100 mg/kg/day and the kidneys of males and females given 100 mg/kg/day. Forestomach changes consisted of very slight hyperplasia and very slight hyperkeratosis of the limiting ridge epithelium in males given 30 or 100 mg/kg/day and females given 100 mg/kg/day and very slight multifocal vacuolization of limiting ridge epithelium in males given 100 mg/kg/day. Less frequently, squamous epithelial hyperplasia, hyperkeratosis, focally extensive

submucosal edema and inflammation extended to other regions of the non-glandular mucosa in a few rats given 100 mg/kg/day. In the glandular stomach, treatment-related changes consisted of 1) very slight, neck mucous cell hyperplasia in males given 30 mg/kg/day and in rats of both sexes given 100 mg/kg/day and 2) a very slight subacute to chronic inflammation in males and females given 100 mg/kg/day. All of these treatment-related gross and histopathological changes in the stomach were interpreted to be localized responses to a repeatedly administered irritant test material at the portal of entry. In males given 30 mg/kg/day, the treatment-related stomach effects, which comprised of very slight squamous hyperplasia and hyperkeratosis of the limiting ridge and the very slight neck mucous cell hyperplasia of the glandular stomach, were interpreted to be non-adverse changes consistent with a localized adaptive response of the stomach mucosa following repeated exposure to the irritant effects of the test material.

Systemic exposure-related changes were confined to the kidney in a few rats given 100 mg/kg/day and consisted of unilateral or bilateral, very slight multifocal hyperplasia and hypertrophy of the medullary tubular epithelium. Although the exact reason is not clear, the kidney effects were likely related to possible regulation of acid-base balance associated with the urinary excretion of the test material and/or its metabolites.

Based on these results, the lowest-observed-adverse-effect level (LOAEL) for systemic toxicity as well as localized portal of entry (stomach) effects was determined to be 100 mg/kg/day in males and females. The no-observed-adverse-effect level (NOAEL) for systemic toxicity and portal of entry effects was 30 mg/kg/day. The NOAEL for reproductive and neurological effects in males and females was 100 mg/kg/day, the highest dose level tested.