Reaction mass of Benzene sulfonic acid, hexadecyl(sulfophenoxy)-,disodium salt and Benzene sulfonic acid, - oxibis[hexadecyl]-, disodium salt

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650- Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. July 2000.

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

405-430-6

EC Name:

-

Cas Number:

65143-89-7

Molecular formula:

UVCB

IUPAC Name:

Reaction mass of Benzene sulfonic acid, hexadecyl(sulfophenoxy)-,disodium salt and Benzene sulfonic acid, - oxibis[hexadecyl]-, disodium salt

Test material form:

other: liquid

Details on test material:

Dowfax Detergent Surfactant
Reference: M00122291
Source: The Dow Chemical Company, Connecticut
Purity: 36.7 ± 0.06%
Purity/Characterization: The solids content of the sample was determined to be 37.5 ± 0.05%, using an oven drying method and duplicate analyses (VanderKamp et al., 2002). The weight percent of sodium chloride and sodium sulfate were determined by ion chromatography to be 0.16 ± 0.02% and 0.57 ± 0.02%, respectively. These values were determined by subtracting the concentrations of sodium chloride and sodium sulfate from the total solids. A purity of 36.7 ± 0.06% was reported for the sample. Infrared spectroscopy was used to confirm the proposed structure of Dowfax 8390 surfactant.
Appearance: Light brown liquid
Molecular Formula: disodium hexadecyldiphenyloxide disulfonate (C28H40S2O7Na2); disodium dihexadecyldiphenyloxide disulfonate (C44H72S2O7Na2)

Description: Dowfax 8390 is a detergent surfactant primarily composed of disodium hexadecyldiphenyloxide disulfonate (15-35%) and disodium dihexadecyldiphenyloxide disulfonate (5-10%). The test material also contained small amounts of sodium sulfate (1.5% maximum) and sodium chloride (< 1%) with the balance comprised of water.

Test animals

Species:

rat

Strain:

other: CD rats (Sprague-Dawley derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Supplier: Charles River Laboratories Inc. (Portage, Michigan)
Age and Weight: Approximately 10-11 weeks of age (females weighing approximately 200 – 250 grams and males weighing approximately 300 – 400 grams) at the start of the study.

Physical and Acclimation
Each animal was evaluated by a laboratory veterinarian or a trained animal/toxicology technician, under the direct supervision of a lab veterinarian to determine their general health status and acceptability for study purposes upon arrival at the laboratory1. The animals were housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately two weeks prior to the start of the study.

Housing
Animals were housed one per cage (prebreeding) or two per cage (one male and one female during breeding) in stainless steel cages after assignment to the study. The room relative humidity and temperature was maintained within a range of 40-70% and 22 ± 3 °C, respectively. A 12-hour light/dark photocycle was maintained for all animal room(s) with lights on at 6:00 a.m. and off at 6:00 p.m., and room air was exchanged approximately 12-15 times/hour. Cages had wire-mesh floors that were suspended above catch pans and contained a feed container and a pressure activated nipple-type-watering system.

Dams were housed one per cage (with their litter) in plastic cages containing ground corn cob nesting material from approximately gestation day (GD) 19 and throughout the lactation phase of the study.

Randomization and Identification
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 placed on study were uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) which were correlated to unique alphanumeric identification numbers. If a transponder stopped functioning or was lost, it was replaced with a new transponder which was correlated with the unique animal number.

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. The results of these analyses indicated no contaminants at levels that would interfere with the conduct of this study or interpretation of the results. Copies of these analyses are maintained at Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

Animal Welfare
In response to the Final Rules amending the U.S. Animal Welfare Act promulgated by the U.S. Department of Agriculture effective October 30, 1989, the Animal Care and Use Activities (ACUA) required for the conduct of this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC determined that the proposed Activities were in full accordance with these Final Rules. The IACUC assigned file numbers DART 01, Neurotox 01, DCO 01, and Animal ID 01 to these Animal Care and Use Activities.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on exposure:

Route and Method of Administration
The test material was administered orally via gavage using a stainless steel feeding needle. Dose volumes for males and females during the premating and mating phases of the study were adjusted based on a weekly body weight. Dose volumes for mated females were adjusted on gestation days 0, 7 14, and 20. For females delivering a litter, dose volume adjustments were made on lactation days 1, 4, and 11. Dose
volumes for females that failed to mate or deliver litters were based on weekly body weights.

Frequency and Duration
Dosing began on September 12, 2002. Males were dosed for two weeks prior to mating, continuing throughout mating, and until necropsy on test day 47. Females were dosed daily for two weeks prior to mating, continuing throughout mating, gestation, lactation, and until the day of necropsy (test day 50 or 54).

Route Justification
Oral gavage is the preferred route of exposure according to OECD Guideline 422.

Dose Levels and Justification
Dose Levels
(mg/kg/day) No. of Rats/Sex/Dose
0 12
25 12
75 12
250 12
TOTAL 96
The high-dose level was based upon body weight, feed consumption, hematology, and prothrombin time data obtained from the initial start of the main study in which males and females were dosed for at least 28 days. The high dose of 250 mg/kg/day was expected to induce some toxic effects, but not death or obvious suffering. The lower dose levels were selected to provide dose response data for any toxicity that may be observed among the high-dose group rats and to establish a no-observable-effect level (NOEL).

Dose Preparation
The test material was administered in an aqueous vehicle (distilled water), such that a dose volume of 4 ml/kg body weight yielded the targeted dose (expressed as actual surfactant, present at 37% in the aqueous sample). Dose solutions for the main study were prepared periodically throughout the dosing period based upon stability analyses performed during the rangefinder portion of this study.

Details on mating procedure:

Breeding of the adults commenced after 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 were detected or a vaginal copulatory plug was observed in situ was considered day 0 of gestation. The sperm or plug-positive (presumed pregnant) females then were separated from the male and returned to their individual cages. If mating did not occur 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
Homogeneity
The low- and high-dose solutions from the first mix of the probe study and the repeat start of the main study were analyzed to confirm homogeneous distribution of the test material concurrent with the start of dosing.

Stability
The stability of the test material was determined during the conduct of the rangefinder and prior to the start of the main study. The targeted concentrations of the samples that were analyzed were 0.25 mg/ml and 187.5 mg/ml.

Concentration Verification
Analyses of all dosing solutions from the first mix of the probe study and the repeat start of the main study were initiated prior to the start of the respective dosing periods using HPLC with ultraviolet detection and external standards to determine actual concentrations.

Duration of treatment / exposure:

The females were dosed once daily for 2 weeks prior to breeding, continuing through breeding (2 weeks), gestation (3 weeks), lactation (4 days) and until the day of necropsy (test day 50 or 54). Females exhibiting active signs of parturition at the time of scheduled gavage were not dosed on that particular day (comment was made on dos ng record). The males were dosed once daily for 2 weeks prior to breeding and continuing through breeding (2 weeks) up until the day of necropsy (test day 47).

Frequency of treatment:

daily

Details on study schedule:

Groups of 12 male and 12 female CD rats were administered the test material 7 days/week by gavage at dose levels of 0 (control), 25, 75, or 250 mg/kg/day. These dose levels represented doses of the actual surfactant present at 37% in the aqueous sample. The females were dosed once daily for 2 weeks prior to breeding, continuing through breeding (2 weeks), gestation (3 weeks), lactation (4 days) and until the day of necropsy (test day 50 or 54). Females exhibiting active signs of parturition at the time of scheduled gavage were not dosed on that particular day (comment was made on dos ng record). The males were dosed once daily for 2 weeks prior to breeding and continuing through breeding (2 weeks) up until the day of necropsy (test day 47). Effects on general toxicity, neurobehavioral activity, clinical pathology, 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. In the offspring, litter size, pup survival, sex, body weight, and the presence of gross external abnormalities were assessed.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 25, 75, or 250 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

12 males and 12 females/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

No additional information available.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Daily Observations
Twice each day a cage-side examination was conducted and to the extent possible the following parameters evaluated: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behavior, moribundity, mortality, and the availability of feed and water.

Moribund animals not expected to survive until the next observation period, and any animals found dead were necropsied on that day. Animals found dead after routine working hours or on weekends or holidays were refrigerated until the next scheduled work day at which time they were necropsied.

Detailed Clinical Observations
Detailed clinical observations (DCO) were conducted pre-exposure, and then weekly throughout the study (gestation days 1, 7, 14, and 20, and lactation day 5 for pregnant females). The DCO were 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 that were recorded categorically or using explicitly defined scales (scored).

Functional Tests
The functional tests included a sensory evaluation, rectal temperature, grip performance,and motor activity. Functional tests were conducted pre-exposure and during the last week of the treatment period. For the females, this took place on post-partum (pp) day 11.

Sensory Evaluation
The sensory evaluation included a test for nociception (responsiveness to tail pinch) and for startle response (responsiveness to sharp noise). The evaluation was conducted in a small clear plastic box.

Rectal Temperature
Rectal temperature was measured by carefully placing a rectal thermistor (Physitemp RET-2, T-type) approximately 4 cm into the rectum for about 15-20 seconds. Temperature was then recorded. The thermistor was validated at 37 °C before and after the study. The instrument was re-calibrated if the validation temperature recordings differ from the reference thermometer by more than ± 0.5 °C.

Grip Performance
Hind-limb grip performance was tested according to the procedure described by Mattsson et al. (1986). Briefly, the observer placed the animal’s forelegs on a bench and the hind-feet were set on a horizontal screen attached to an electronic strain gauge (Chatillon, Greensboro, North Carolina; model DFGHS with a 1-kg load cell). The observer then smoothly but firmly pulled backward on the tail until the animal’s grip on the screen was broken. An electronic strain gauge was used to record the animal’s resistance to the pull in grams. The average of three trials was used for statistical analysis. Forelimb grip performance was similarly tested. In this application, a bench was not used, and the rats were placed so that the forefeet were on the screen and the hind-feet were suspended approximately 10 cm above the smooth horizontal plastic surface.

Instrument Validation
A standard 500 g weight attached to a fine-gauge wire was suspended from the load cell (weight was measured from the load cell in grams). A 1% tolerance (i.e. 500 ± 5 grams) was acceptable and was checked just before and just after testing.

Motor Activity
An automated system was used for motor activity (MA) data collection. No entry into the MA test room was allowed during the testing period. Each test session consisted of ten 8 minute epochs, totaling 80 minutes of testing per animal per test session. This duration was chosen based on the results of a validation study indicating that performance of control animals approached asymptote in 70-80 minutes in Sprague Dawley rats (Marable and Andrus, 2001). Activity counts for each epoch were recorded.

Motor Activity Cage Calibration
Cages used for testing were calibrated prior to testing each day. Calibration was performed with a rod attached to a rotary motor that broke the infrared beam at a constant speed. The duration of each beam break was calculated to ensure equivalence across chambers.

Motor Activity Cage Allocation
The rats were allocated to the motor activity cages in such a way the counterbalancing of treatment groups and sexes across cages and test times was maximized.

Body Weights
All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Males 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 days 0, 7, 14, and 20. During lactation females were weighed on days 1, 4, and 11. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. Statistical analysis of body weights and body weight gains during gestation was performed using data collected on days 0, 7, 14, and 20. Statistical analysis of body weights and body weight gains during the postpartum period were performed using data collected on days 1, 4, and 11.

Feed Consumption
Feed consumption was measured weekly during the two week pre-breeding period by weighing feed crocks at the start and end of a measurement cycle. During breeding, feed consumption was not be measured in males or females due to co-housing. Following breeding, feed consumption was not measured for males. During gestation, feed consumption was measured for females on days 0- 7, 7-14, and 14-20. During the postpartum period, feed consumption was measured on days 1-4 and 4-11. Feed consumption was not measured for females that failed to mate or failed to 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
On the day prior to the scheduled necropsy, all males and females in each dose group were fasted overnight. At necropsy, the animals were anesthetized with CO2, and then blood samples were collected from the orbital sinus. Blood samples were not obtained from females that failed to deliver a litter. Blood samples were not obtained from males or females that died or were sacrificed moribund prior to the scheduled necropsy.

Coagulation
Sample Preparation
Blood samples were collected in sodium citrate tubes, centrifuged and plasma collected and assayed using an ACL9000 (Instrumentation Laboratory Lexington, Massachusetts).

Assay
Prothrombin time (PT)

Hematology
Sample Preparation
Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA) and smears were prepared, stained with Wright’s stain and archived. Hematologic parameters were assayed using a Technicon H·1E Hematology Analyzer (Bayer Corporation, Tarrytown, New York).

Assays
Hematocrit (HCT)
Hemoglobin (HgB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Platelet (PLAT) count
Differential WBC count
Red blood cell indices (MCH, MCV and MCHC)

Clinical Chemistry
Sample Preparation
Serum was separated from cells as soon as possible following blood collection. Serum parameters were measured using a Hitachi 914 Clinical Chemistry Analyzer (Boehringer-Mannheim, Indianapolis, Indiana).

Enzyme Activities of:
Alkaline phosphatase (AP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)

Concentrations of:
Albumin (ALB)
Cholesterol (CHOL)
Creatinine (CREAT)
Electrolytes (Na, K, PO4, Cl and Ca)
Glucose (GLU)
Total bilirubin (TBILI)
Total protein (TP)
Urea nitrogen (UN)

Urinalysis
A timed urine volume was obtained from all male rats in each dose group (nonfasted) during the week prior to the scheduled necropsy. Animals were
housed in metabolism cages and urine collected overnight (approximately 16 hours).

Assays
Color, appearance, specific gravity (refractometer) and urine volume. Semiquantitative analysis (MultistixÒ Reagent Strips, Bayer Corporation, Elkhardt, Indiana on the Clinitek 200+) of:
pH
Bilirubin
Glucose
Proteins
Ketones
Blood
Urobilinogen

Urine was also collected by manual compression of the bladder. The urine was pooled for each group, and the microsediment was characterized
microscopically.

Oestrous cyclicity (parental animals):

Not analyzed

Sperm parameters (parental animals):

Not analyzed

Litter observations:

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

Postmortem examinations (parental animals):

Necropsy
A complete necropsy of all the adults was performed. All males were necropsied on test day 47. Females that delivered litters were necropsied on or shortly after postpartum day 12 (test days 50 and 54). Dosing continued until the day prior to sacrifice. Females that did not deliver a litter were necropsied at least 24 days after the last day of the mating period. Both males and females were fasted overnight prior to necropsy. Fasted adult rats submitted alive for a necropsy were anesthetized by CO2 vapors, weighed, blood samples were collected, 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 nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphatebuffered 10% formalin using a hand-held syringe and blunt needle. 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 uteri of all females were examined, and the number of implantation sites were recorded. The uteri of females that did not deliver litters were stained with a 10% solution of sodium sulfide in order to verify pregnancy status (Kopf et al., 1964).

The following tissues were trimmed and weighed: testes, epididymides, ovaries, liver, kidneys, adrenals, thymus, spleen, brain, thyroid/parathyroid (after fixation) and heart. The organ to body weight ratios were calculated.

Representative samples of selected tissues were collected and preserved in neutral, phosphate-buffered 10% formalin, except that the testes and epididymides were preserved by immersion in Bouin’s fixative. Similar necropsy procedures were followed for animals found dead or moribund with the exception that terminal body weights and organ weights were not taken. Transponders were removed and placed in jars with the tissues.

Tissues Collected and Preserved at Necropsy
ADRENALS (2)* KIDNEYS (2)* PROSTATE (1)*
AORTA (1) LACRIMAL/HARDERIAN GLANDS (2) RECTUM (1)*
AUDITORY SEBACEOUS GLANDS (0) LARYNX (1) SALIVARY GLANDS (1)
BONE (1) (INCLUDING JOINT)(1) LIVER (3)* SEMINAL VESICLES (2)*
BONE MARROW (1)* LUNGS (5)* SKELETAL MUSCLE (1)
BRAIN (CEREBRUM, BRAINSTEM, CEREBELLUM) (3)* MAMMARY GLAND - FEMALES ONLY(1)* SKIN AND SUBCUTIS (1)
CECUM (1)* MEDIASTINAL LYMPH NODE (1)* SPINAL CORD (CERVICAL, THORACIC, LUMBAR) (3)*
CERVIX (1)* MEDIASTINAL TISSUES (1) SPLEEN (1)*
COAGULATING GLANDS (2)* MESENTERIC LYMPH NODE (1)* STOMACH (1)*
COLON (1)* MESENTERIC TISSUES (1) TESTES (2)*
CRANIAL NERVE – OPTIC (2) NASAL TISSUES/PHARYNX (2) THYMUS (1)*
DUODENUM (1) ORAL TISSUES (2) THYROID GLAND (1)*
EPIDIDYMIDES (2)* OVARIES (2)* TONGUE (0)
ESOPHAGUS (1) OVIDUCTS (2)* TRACHEA (1)*
EYES (2) PANCREAS (1) URINARY BLADDER (1)*
GROSS LESIONS (1)* PARATHYROID GLANDS (1) UTERUS (4)*
HEART (1)* PERIPHERAL NERVE -TIBIAL(1)* VAGINA (1)*
ILEUM (WITH PEYER’S PATCH)(1)* PITUITARY (1)*
JEJUNUM (1)*
The number in parenthesis represents the minimum number of sections examined histopathologically.
* Tissues selected for histopathological examination.

Histopathology
Histologic examination of the tissues and tissues with relevant gross lesions was conducted on all adult rats from the control and high-dose groups and all rats found dead or sacrificed moribund. 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 mm and stained with modified periodic acid- Schiffs-hematoxylin. The presence and integrity of the 14 stages of spermatogenesis were 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, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis. Examination of tissues from the low- and mid-dose groups was limited to the lungs and trachea (to confirm the presence of alterations associated with the oral gavage procedure) 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.

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 would not be 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 may have been 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 would not be 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 may be life threatening.

Postmortem examinations (offspring):

All pups surviving to lactation day 4 were euthanized by oral administration of sodium pentobarital solution, examined for gross external alterations, and then discarded. Any pups found dead or euthanized in moribund condition were examined to the extent possible and discarded.

Statistics:

Descriptive statistics only (means and standard deviations) were reported for RBC indices, and WBC differential counts. Parental body weights and parental body weight gains, litter mean body weights, feed consumption, urine volume, urine specific gravity, coagulation, clinical chemistry data, appropriate hematologic data, and organ weights (absolute and relative) were first evaluated by Bartlett's test (a = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at a = 0.05, a Dunnett's test (a = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (a = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Feed consumption values were excluded from analysis if the feed had been spilled or scratched.

Statistics continued below

Reproductive indices:

Calculation of Reproductive Indices
Reproductive indices were calculated for all dose levels as follows:
· Female mating index = (No. females with evidence of mating/No. paired) x 100
· Male mating index = (No. males with evidence of mating/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 viable litter/No. females delivering a litter) x 100
· Gestation survival index = percentage of delivered pups alive at birth
· Post-implantation loss = (No. implants – No. viable 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

Offspring viability indices:

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:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

Analytical Chemistry
Analyses of all dosing solutions from the first mix of the initial start of the main study revealed mean concentrations of Dowfax 8390 Surfactant ranging from 101% to 111% of targeted concentrations. Results from the analysis of the solutions mixed for the repeat start of the study indicated mean concentrations of Dowfax 8390 surfactant ranging from 97.3% to 99.2% of targeted concentrations. Analysis of aliquots for the low and high-dose solutions from both mixes indicated that the test material was homogeneously distributed. Dowfax 8390 Surfactant was stable in distilled water for at least 17 days.

Mortality
A 25 mg/kg/day group male (4395) was found dead on test day 16. Observations noted on this male prior to spontaneous death included struggle during dosing, perinasal/perioral soiling (clear), and labored respiration with mouth breathing subsequent to dosing. At necropsy, congestion was noted in the liver, lungs, and kidneys. Generalized edema was also noted in the lungs. Subsequent histopathologic observations made for this male were consistent with a probable cause of death due to a gavage dosing complication. Two 75 mg/kg/day females died prior to scheduled necropsy. Dam 5632 exhibited red vulvar discharge on gestation day 0 and subsequently died on gestation day 15. Upon gross examination, this dam had blood on the nares, froth in the trachea, serosanguineous hydrothorax, mottled lungs, and a pregnant uterus. Histologic observations recorded for this female included congestion in the adrenal glands and liver, as well as alveolar and perivascular edema in the lungs which was suggestive of a gavage dosing error as the cause of death. Dam 5634 was found dead on lactation day 5, after having successfully cared for her litter through lactation day 4. This rat exhibited labored respiration with mouth-breathing just prior to death. At gross examination, the lungs contained froth and were mottled, consistent with aspiration of test material. The probable cause of death for this dam was a gavage dosing complication.

Clinical Observations
In the 250 mg/kg/day group, increased incidences of various respiratory difficulties, clear perioral soiling (both sexes), clear perinasal soiling (males only), red perinasal soiling (males only), and decreased/soft feces (males only). The clear perinasal soiling was temporally correlated with clear perioral soiling. The clear perioral/perinasal soiling appeared to be a salivation response which was transient, tending to occur sporadically and often just prior to dosing. Salivation may have been related to the physical properties of the compound, but was not considered a toxicological response. At 75 mg/kg/day, similar clear perioral and/or red perinasal soiling (males only) and respiratory difficulties (both sexes) occurred at slightly higher frequencies than controls. In the 25 mg/kg/day group, there was a slight increase in the number of males exhibiting red perinasal and/or clear perioral soiling. The respiratory symptoms noted in a single male in this group were related to a gavage dosing complication. In all groups, most animals exhibiting red perinasal soiling did so for only one day out of the entire study period, with this finding being noted no more than four times in any given animal. Therefore, this minor finding, which was also seen in control animals, was not considered toxicologically significant. Also, the respiratory symptoms noted in all groups appeared to be iatrogenic effects related to aspiration of small amounts of the surfactant test material (see Histopathology section for further discussion). Other clinical observations were not considered treatment-related due to their low incidence, lack of a dose-response and/or transient nature.

Detailed Clinical Observations
Examinations performed on all animals weekly throughout the study revealed no treatment-related or statistically significant findings.

Feed Consumption
There were no significant differences in the amount of feed consumed by any of the treated groups when compared
to their respective controls throughout the premating, gestation, or lactation periods.

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

Reproductive Indices, Pup Survival, and Sex Ratio
There were no treatment related effects at any dose level on any of the reproductive parameters evaluated in this study. Values for pup survival, sex ratio, gestation length, time to mating, and postimplantation loss were comparable to control values.

Functional Tests
Sensory Evaluation
Evaluations performed on males and females at study termination revealed no treatment-related findings.

Rectal Temperature
There was no treatment effect in males (p = 0.8047). In females, the treatment-by-baseline interaction was statistically significant, thereby violating the homogeneity of slopes assumption of the analysis of covariance. As per protocol, the analysis of covariance was replaced by ANOVAs at the two time points in females. There were no group effects either under baseline conditions (p = 0.8158) or after treatment (p = 0.9180).

Grip Performance
There were no treatment effects on hindlimb grip performance either in males (p = 0.2859) or females (p = 0.9924). Similarly, there were no treatment effects on forelimb grip performance either in males (p = 0.5950) or females (p = 0.4296).

Motor Activity
Treatment did not affect motor activity total counts (treatment-by-time interaction) either in males (p = 0.2725) or in females (p = 0.2082). Similarly, the distribution of the motor activity counts within session (treatment x time x epoch interaction) was not affected by treatment either in males
(p = 0.7047) or in females (p = 0.8919).

Clinical Pathology
Coagulation
Statistically significant increases in prothrombin times were observed for males given 75 (19% increase) or 250 (35% increase) mg/kg/day of Dowfax 8390 relative to controls. These increases in prothrombin time had no apparent functional impact, as evidenced by the lack of any complications of parturition or episodes of unusual bleeding. There was no effect on prothrombin times for males given 25 mg/kg/day. There were no treatment-related effects on prothrombin times for females given 25, 75, or 250 mg/kg/day. A statistically significant decrease in prothrombin time identified for females given 250 mg/kg/day was considered spurious as it was not consistent with the effect noted in males (increased prothrombin time rather than decreased) and similar increases in prothrombin time were not seen in females administered higher doses in the initial phase of this study.

Prothrombin Time – Statistically Identified Differences
Historical Control
Data
Parameter Study 1a 0 MKD 25 MKD 75 MKD 250 MKD
Prothrombin
Time(males) 12.1 12.4 13.2 14.8* a 16.7*a

Prothrombin
Time
(female)
11.8 12.0 12.0 11.7 11.3*
a Inhalation study conducted in 2003
* Statistically different from control mean (alpha = 0.05).
a- indicates treatment related effect.

Hematology
There were no treatment-related hematologic effects at any dose level.

Clinical Chemistry
Males and females given 250 mg/kg/day had statistically identified treatment-related increases in mean serum ALT activity. Statistically identified
increase in the mean AST activity was also seen in females given 250 mg/kg/day and although this value falls within the historical control range, it was interpreted to be treatment-related due to a concomitant increase in serum ALT levels. The increase in these ‘leakage’ enzymes, particularly ALT, is indicative of hepatocellular injury. However, there was no significant histologic evidence of hepatocellular injury.

Minimal, but statistically identified decreases in blood urea nitrogen were noted in males given 75 or 250 mg/kg/day. These were considered unrelated to treatment due to lack of a clear dose-response relationship and also because they were within historical control range. Toxicologically significant alterations are normally associated with increases in blood urea nitrogen levels.

A minimal, but statistically identified decrease in total serum protein was noted in males given 250 mg/kg/day. This minimal decrease was within the historical control range and hence, interpreted to be not treatment-related.

Clinical Chemistry – Statistically Identified Differences (Males)
Historical Control Data
Parameter Study 1a Study 2a Study 3b 0 MKD 25 MKD 75 MKD 250 MKD
Urea
Nitrogen
(mg/dl) 15 13 14 17 16 14* 15*
ALT (u/l) 29 33 32 47 40 40 83*a
Total Protein
(g/dl) 6.4 6.3 6.2 6.6 6.5 6.4 6.3*
a Inhalation studies
b Oral gavage study (The two inhalation studies and the one oral gavage study were done since 2000)
* Statistically different from control mean (alpha = 0.05)
a-indicates treatment-related effect.

Clinical Chemistry – Statistically Identified Differences (Females)
Historical Control Data
Parameter Study 1a Study 2a Study 3b 0 MKD 25 MKD 75 MKD 250 MKD
ALT (u/l) 36 63 27 30 31 33 69*a
AST (u/l) 94 120 107 77 76 78 96*a
a Inhalation studies
b Oral gavage study (The two inhalation studies and the one oral gavage study were done since 2000)
* Statistically different from control mean (alpha = 0.05)
a- indicates treatment-related effect.

Urinalysis
Relative to the controls, males given 250 mg/kg/day had slightly lower urine volume and slightly higher specific gravity. The lower urinary volume of the males given 250 mg/kg/day was higher than that of the historical controls and the higher specific gravity was also within the historical control range and hence these alterations were interpreted to be not treatment-related. The higher specific gravity and lower urine volumes support the premise that the kidneys were functioning at a higher level than the controls.

Urine samples from males given > or = 25 mg/kg/day showed a higher incidence of alkalinuria with a urinary pH of 8.5 or > or = 9.0 for most of the animals. However, in the absence of any significant serum electrolyte alterations and lack of evidence of histological changes in the kidneys, the higher incidence of alkalinuria may be due to the urinary excretion of the test material and/or its metabolites.

Five out of twelve males given 250 mg/kg/day had bilirubinuria (1+) as compared to one out of twelve in the controls. This increase in the incidence of bilirubinuria in this treatment group was interpreted to be secondary to the increase in urine specific gravity in this group. All the five rats with 1+ bilirubinuria had a urine specific gravity of 1.064 or above compared to the control mean of 1.046. Secondly an increased incidence of 1+ bilirubinuria has also been noted in the historical controls and therefore this change was interpreted to be not treatment-related. Two of twelve males (animal identification numbers 5596 and 5599) given 250 mg/kg/day had blood (4+) in the urine. The reason for the hematuria in these two animals was not apparent as their individual prothrombin times (13.6 and 13.9 seconds respectively) were within the range of the concurrent controls (11.6 – 14.5
seconds) and there were no gross or histological indications of hemorrhage, inflammation, or trauma in the urinary tract examined. Pooled urine sediment from this treatment group had erythrocytes that were too numerous to count.

Urinalysis (Males)
Historical Control Data
Parameter Study 1a Study 2b 0 MKD 25 MKD 75 MKD 250 MKD
Volume
(ml) 7.2 9.7 14.3 14.1 13.1 10.3
Specific
Gravity 1.067 1.057 1.046 1.043 1.049 1.059
pH 7.0 (3) 7.0 (1) 7.0 (1) 8.0 (2) 8.5 (10)a 7.5 (1)
7.5 (4) 7.5 (3) 7.5 (1) 8.5 (6)a > or = 9 (2)a 8.0 (1)
8.0 (2) 8.0 (1) 8.0 (7) > or = 9 (3)a 8.5 (1)
8.5 (3) 8.5 (3) 8.5 (3) > or = 9 (9) a
> or = 9 (3)

Bilirubin Neg (7) Neg (9) Neg (11) Neg (10) Neg (11) Neg (7)
+ (5) + (2) + (1) + (1) + (1) + (5)

Blood Neg (10) Neg (10) Neg (11) Neg (11) Neg (12) Neg (10)
+ (2) + (1) + (1)
++++ (2)
a Inhalation study
b Oral gavage study (The inhalation and the oral gavage studies were done since 2000)
a- indicates treatment-related effect.

Organ Weights
There were no treatment-related differences in any of the organ weights for male or females at any dose level. A slight, but statistically identified increase in relative thyroid weight for females given 75 mg/kg/day was not considered biologically significant as absolute weight was not similarly affected and no effect was seen in females at the higher dose of 250 mg/kg/day.

Gross Pathology
There were no treatment-related gross pathological findings.

As mentioned previously (Mortality section), there were three spontaneous deaths during the course of this study; two females given 75 mg/kg/day and one male given 25 mg/kg/day. These animals variably showed hydrothorax, mottled lungs, froth in trachea or lungs, generalized edema of lungs and congestion of liver, lungs and kidneys. These lesions were consistent with inadvertent gavage complication which was the cause of death.

In some females, very slight to slight alopecia was seen across all treatment groups including the controls. These areas of alopecia were variably distributed in areas such as the abdomen, back, forefoot, forelimb, and shoulder. Two males given 25 mg/kg/day showed very slight, unilateral (right side) alopecia, one on the right forelimb and the other on right shoulder. One male given 250 mg/kg/day showed very slight bilateral alopecia on the forelimbs. Although no control males were found with such alopecia, these isolated cases with no dose-response relationship, coupled with the observation of alopecia in females across all treatment groups including the controls, indicated that these observations were not treatment-related.

Gross observations in the lungs such as, generalized edema, pale/dark focus, froth, and mottled lungs were consistent with gavage complications observed at scheduled necropsy or in spontaneously dead animals. All other pathologic observations listed in the table and not discussed herein were interpreted to be spontaneous changes not associated with the exposure to Dowfax 8390.

Histopathology
There were no treatment-related histopathological changes attributed to the direct systemic toxicity of Dowfax 8390.

However, the lungs and trachea of some animals of both sexes given 25, 75, or 250 mg/kg/day had histopathological changes consistent with aspiration into the respiratory tract following reflux of the test material (possibly due to the surfactant properties of the test material) that may have occurred during the course of repeated oral gavage and/or inadvertent gavage errors. These changes variably consisted of very slight, slight or moderate, multifocal, chronic/chronic-active inflammation of the bronchi, bronchioles, and the alveoli. Very slight to slight perivascular eosinophilic inflammation was seen in the lungs of some males and females, with or without inflammatory changes in the airways. The perivascular eosinophilic inflammatory response was interpreted to be a part of the inflammatory reaction triggered by the inadvertent entry of Dowfax 8390 into the respiratory tract during the process of repeated oral gavage. A very slight degree of perivascular eosinophilic inflammation was, however, seen in one control female, the reason for which was not apparent. All these changes were random in distribution and did not involve all the lung lobes. Other changes indicative of aspiration of the test material into the respiratory tract were:
1) The occasional presence of small quantities of a homogenous eosinophilic material (consistent with a foreign material) in the alveoli of some of the affected lungs;
2) Very slight to slight diffuse regenerative hyperplasia of the tracheal epithelium in two males given 75 mg/kg/day and in one male given 250 mg/day;
3) Slight subacute to chronic focal inflammation of the trachea of one female given 250 mg/kg/day; and
4) Slight chronic focal inflammation of the visceral pleura of one female given 250 mg/kg/day.

These histopathologic changes in the lungs and trachea were interpreted to be an artifact of test material delivery and not related to the absorption and systemic toxicity of Dowfax 8390 for the following reasons:
1. Isolated occurrences across all groups treated with Dowfax 8390
2. No dose-response relationship in either incidence or severity of the lesions
3. Lack of uniform distribution and lack of involvement of all lobes of the lung as would be expected with systemic toxicity.

Gavage Induced Lesions of the Lungs and Trachea
Sex Males Females
Dose (MKD) 0 25 75 250 0 25 75 250
Number Examined 12 12 12 12 12 12 12 12

Lung
Inflammation; chronic; bronchibronchiole;
bronchiolo-alveolar;
multifocal Very slight 0 0 0 0 0 0 0 1
Slight 0 0 1 0 0 0 0 1
Moderate 0 0 0 2 0 0 0 0
Inflammation; chronic active;
bronchi-bronchiole; bronchioloalveolar;
multifocal Very slight 0 0 0 1 0 0 0 0
Slight 0 0 1 0 0 0 0 1
Moderate 0 0 1 0 0 0 0 0
Inflammation; eosinophilic;
perivascular; multifocal Very slight 0 2 1 1 1 1 0 1
Slight 0 0 1 1 0 0 1 1
Inflammation; chronic; pleura; focal
Slight 0 0 0 1 0 0 0 0


Trachea
Hyperplasia;regenerative,
epithelium; diffuse Very slight 0 0 1 0 0 0 0 0
Slight 0 0 1 1 0 0 0 0
Inflammation; subacute to chronic;
lamina propria; submucosa; focal
Slight 0 0 0 0 0 0 0 1

Skin lesions were observed in males and females across all treatment groups including controls, but slightly more so in the females. Histopathologic changes variably included very slight to slight atrophy of hair follicles (hair follicles in telogen or resting phase), very slight, slight or moderate epidermal hyperplasia and hyperkeratosis, and very slight, slight or moderate chronic focal/multifocal inflammation of the dermis. These skin lesions were non-specific and were attributed to scratching and/or excessive grooming and hence not treatment-related.

All histopathologic observations including those discussed above were interpreted to be either iatrogenic (lung and tracheal lesions) or spontaneous changes (all other organs) unassociated with systemic toxicity of Dowfax 8390.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

Reproductive Indices, Pup Survival, and Sex Ratio
There were no treatment related effects at any dose level on any of the reproductive parameters evaluated in this study. Values for pup survival, sex ratio, gestation length, time to mating, and postimplantation loss were comparable to control values (Table 1).

Litter Observations
Observations recorded in the offspring occurred at low frequency and bore no relationship to treatment. There were no visible external morphologic alterations noted in any of the offspring.

Litter Size and Pup Body Weights
There were no treatment-related effects on litter size or pup body weights.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects were noted in the F1 generation at the highest dose level examined.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1 DOWFAX 8390 SURFACTANT: A COMBINED REPEATED DOSE TOXICITY STUDY WITH THE REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST IN CD RATS

Reproduction Indices and Pup Survival

Dose mg/kg/day

0

25

75

250

Number of Males	12	11*	12	12
Number of Females	12	12	12	12
Male Mating Index, %^A	100 (12/12)	100 (11/11)	100 (12/12)	100 (12/12)
Female Mating Index, %^B	100 (12/12)	100 (11/11)	100 (12/12)	100 (12/12)
Male Conception Index, %^C	91.7 (11/12)	81.8 (9/11)	91.7 (11/12)	83.3 (10/12)
Female Conception Index, %^D	91.7 (11/12)	83.3 (10/12)	91.7 (11/12)	83.3 (10/12)
Male Fertility Index, %^E	91.7 (11/12)	81.8 (9/11)	91.7 (11/12)	83.3 (10/12)
Female Fertility Index, %^F	91.7 (11/12)	83.3 (10/12)	91.7 (11/12)**	83.3 (10/12)
Gestation Index, %^G	100.0 (11/11)	100.0 (10/10)	100.0 (10/10)	100.0 (10/10
Gestation Survival Index,%^H	100 (166/166)	97.9 (139/142)	98.4 (127/129)	98.7 (149/151)
Day 1 Survival Index, %^I	100 (166/166)	100 (139/139)	96.9 (123/127)	98.7 (147/149)
Day 4 Survival Index, %^I	98.8 (164/166)	99.3 (138/139)	96.1 (122/127)	97.3 (145/149)
Postimplantation Loss, %^J	5.69 + 6.00	9.07 + 10.55	12.61 + 13.13	7.48 + 10.647
Sex Ratio on Day 1 Male:Female	47:53	49:51***	53:47	51:49
Gestation Length (Days)	21.6 + 0.5	21.6 + 0.5	21.8 + 0.6	21.6 + 0.5
Time to Mating (Days)	2.7 + 1.7	2.1 + 1.1	1.8 + 1.1	2.2 + 1.2

* MALE 5569 DIED EARLY AFTER TWO DAYS OF COHOUSING WITH FEMALE 5617 FOR BREEDING. FEMALE WAS GIVEN TO NEXT AVAILABLE MALE AND MALE 5569 WAS EXCLUDED FROM CALCULATIONS OF MALE REPRODUCTIVE INDICES.

** FEMALE 5632 DIED DURING GESTATION, BUT WAS FOUND TO BE PREGNANT WITH NORMALLY DEVELOPED FETUSES AND WAS THEREFORE INCLUDED IN THE CALCULATION OF FEMALE FERTILITY.

*** TABLE 24 OF THE REPORT ERRONEOUSLY STATES THE SEX RATIO AS 49:41. FOLLOWING REANALYSIS OF THE DATA, THE SEX RATIO WAS CHANGED TO 49:51..

^A # MALES WHICH MATED RESULTING IN A SPERM + VAGINAL LAVAGE OR PREGNANT/TOTAL # MALES AND FEMALES COHOUSED X 100%.

^B # FEMALES WHICH WITH A SPERM + VAGINAL LAVAGE OR PREGNANT WITHOUT ADITIONAL EVIDENCE OF MATING/#FEMALES COHOUSED WITH MALES X 100%.

^C # MALES SIRED A LITTER/# MALES MATED X 100%.

^D # FEMALES DELIVERING A LITTER/# FEMALES MATED X 100%.

^E # MALES WHICH SIRED A LITTER/# MALES AND # FEMALES COHOUSED X 100%.

^F # FEMALES DELIVERING A LITTER/# FEMALES COHOUSED WITH MALES X 100%.

^G # FEMALES DELIVERING A LIVE LITTER/# FEMALES DELIVERING A LITTER X 100%.

^H PERCENTAGE OF NEWBORN PUPS THAT WERE ALIVE AT BIRTH

^I # OF LIVE PUPS ON DAY 1 OR 4 /# OF LIVE PUPS ON DAY 0 X 100%.

^J MEAN PERCENT/LITTER (CALCULATED AS [NO. IMPLANTS – NO. VIABLE OFFSPRING]/NO IMPLANTS) X 100

THERE WERE NO STATISTICALLY IDENTIFIED DIFFERENCES FROM THE CONTROL MEAN AT ALPHA = 0.05.

Applicant's summary and conclusion

Conclusions:

Gavage administration of 250 mg/kg/day of DOWFAX 8390 resulted in increased incidences of soft/decreased feces (males only), accompanied by slightly increased prothrombin times (males only), increased serum ALT, and increased serum AST (females only) levels. At 75 mg/kg/day, prothrombin times were increased in males only. No toxicologically significant effects occurred in the 25 mg/kg/day group. Based on histopathological examination, a number of other clinical signs including transient, sporadic incidences of perioral and/or perinasal soiling, and various respiratory symptoms were considered the result of gavage related aspiration of the surfactant test material. Urinalysis (conducted in males only) revealed a slight increase in urine pH at all dose levels thought to be associated with the properties of the test material and/or its metabolites, but with no toxicological sequelae. There were no treatment-related effects on body weights, body weight gains, feed consumption, detailed clinical observations, neurological end points, reproductive/developmental end points, organ weights, or histopathology at any dose level tested. Based on these data, the no-observable-adverse effect level (NOAEL) for general toxicity was 25 mg/kg/day, while 250 mg/kg/day was a no-observable-effect level (NOEL) for reproductive and neurological effects.

Executive summary:

This study evaluated DOWFAX 8390 surfactant in the OECD 422 study design. Dose levels were selected on the basis of a 14-day range-finding study and an initial OECD 422 study which was terminated early due to unexpected morbidity/mortality (details of both preliminary studies contained in methods section). In the main OECD 422 study which followed, groups of 12 male and 12 female CD rats were administered DOWFAX 8390 surfactant 7 days/week, by gavage at dose levels of 0 (control), 25, 75, and 250 mg/kg/day. Female rats were dosed once daily for 2 weeks prior to breeding, through breeding (2 weeks), gestation (3 weeks), lactation (4 days), and until the day of necropsy (test day 50 or 54). The males were dosed for 2 weeks prior to breeding and continuing through breeding (2 weeks) up until necropsy (test day 47). Effects on reproductive and neurological function as well as general toxicity 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 abnormalities were also assessed.

Gavage administration of 250 mg/kg/day of DOWFAX 8390 resulted in increased incidences of soft/decreased feces (males only), accompanied by slightly increased prothrombin times (males only), increased serum ALT, and increased serum AST (females only) levels. At 75 mg/kg/day, prothrombin times were increased in males only. No toxicologically significant effects occurred in the 25 mg/kg/day group. Based on histopathological examination, a number of other clinical signs including transient, sporadic incidences of perioral and/or perinasal soiling, and various respiratory symptoms were considered the result of gavage related aspiration of the surfactant test material. Urinalysis (conducted in males only) revealed a slight increase in urine pH at all dose levels thought to be associated with the properties of the test material and/or its metabolites, but with no toxicological sequelae. There were no treatment-related effects on body weights, body weight gains, feed consumption, detailed clinical observations, neurological end points, reproductive/developmental end points, organ weights, or histopathology at any dose level tested. Based on these data, the no observable- adverse-effect level (NOAEL) for general toxicity was 25 mg/kg/day, while 250 mg/kg/day was a no-observable-effect level (NOEL) for reproductive and neurological effects.