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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

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

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:

other: OECD 422

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

Deviations:

not specified

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)

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.

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.

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.

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

Duration of test:

47 days for males and 50-54 days for females

No. of animals per sex per dose:

12 males and 12 females/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

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 dosing 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.

Examinations

Maternal 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.

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)

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 thr

Ovaries and uterine content:

Ovaries and uterine content examined as part of the gross and histopathologic examination.

Fetal examinations:

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.

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

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

Historical control data:

Historical control data from OECD 422 studies was used in the interpretation of study results when necessary.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
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.

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 (further details provided in the repeated dose section)

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 (maternal animals)

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

clinical biochemistry

clinical signs

haematology

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
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 (fetuses)

Dose descriptor:

NOEL

Effect level:

250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

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

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%.

^HPERCENTAGE 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%.

^JMEAN 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 this report). 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.