4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08th March 2013 - 17th September 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

with 14 day recovery

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

1

Reference substance name:

4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

EC Number:

299-434-3

EC Name:

4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

Cas Number:

93882-40-7

Molecular formula:

C48H86O8S

IUPAC Name:

2-octadecenylsuccinic anhydride, thiodiethanol esterification products

Test material form:

solid

Remarks:

Waxy Solid

Specific details on test material used for the study:

Lot No: Batch 30038-158
Purity: 100%
Expiration Date: 31 Jan 2014
Description: Brown waxy solid
Storage Condition: Room Temperature, protected from light

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

NTac:SD

Details on species / strain selection:

The Sprague Dawley rat is an acceptable species and strain for assessing toxicity.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing
Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility, following the specifications recommended in the most current version of The Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington, D.C.). Animals were housed in an environmentally controlled room with continuous recording of room temperatures of 69-75¿F and relative humidity of 30-70%, with a 12-hour light/12-hour dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour. Animals were individually housed, upon receipt and during acclimation and following test animal selection, in polycarbonate cages. Sani-Chip Hardwood bedding was used to absorb liquids. Bedding analyses and the lot number and/or milling date are on file at the laboratory there were no contaminants that would have an effect on the results of the study. Cages and feeders were changed at least once weekly. Animals were transferred to clean racks at least once every other week. Access to the Animal Facility was dictated by Standard Operating Procedures (SOPs) and was restricted to authorized personnel only.

Husbandry
The animals were provided ad libitum access to drinking water via an automatic watering system from arrival until termination or removal from the study. Animals were also given supplemental water, using Napa NectarTM (Systems Engineering, Napa, CA), when needed. The water meets U.S. EPA drinking water standards (documentation on file at the laboratory). The animals were provided Harlan TEKLAD Global Diet #2018CM (Certified 18% Protein Rodent Diet, Harlan TEKLAD, Madison, WI) in meal form, in stainless steel rodent feeders, ad libitum from arrival until termination or removal from the study. The lot number and/or milling date were recorded in the raw data. Annual water analyses and individual feed lot analyses (on file at laboratory) did not reveal any contaminants that would have an effect on the results of the study.
The animals were acclimated for 13 and 15 days, males and females, respectively, prior to the first test and/or control article administration. Animals were observed daily for signs of illness or death and any unusual observations or deaths were reported to the Study Director and/or Veterinarian. Prior to randomization, the Veterinarian examined and approved all the animals for study use. All animals assigned to the study appeared normal on Day 1, prior to dosing.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Animals were dosed once daily via oral gavage for 28 consecutive days, as indicated above, beginning on Day 1. The dose volume (Groups 1-4) was 10.0 mL/kg body weight.

Vehicle:

corn oil

Details on oral exposure:

Individual dose volumes were calculated based on the animal’s most recently recorded body weight. Formulations were stirred at least 30 minutes prior to and during dosing.

In addition, on days of FOB evaluations, dosing was staggered during the day for animals being evaluated to allow sufficient time for completion of the evaluations. Formulation container (dose pot) correlation and check was recorded manually on days of FOB evaluation, if needed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Aliquots of dosing formulations prepared for the 28-day study were taken and stored at 2 to 8°C, protected from light, in small volume amber glass containers. Analysis of duplicate samples for concentration, homogeneity and stability (room temperature and refrigerated) were performed by a 3rd party laboratory, using methods for sulfur analysis (%S-OES).

Duration of treatment / exposure:

After completion of 28 days of dosing, 10 rats/sex/dose in Groups 1 to 4 were sacrificed on Day 29. The remaining 5 rats/sex in Groups 1 and 4 entered a 14-day recovery period without dosing and were sacrificed on Day 43.

Frequency of treatment:

Once daily via oral gavage.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15 animals/sex

Control animals:

yes, concurrent vehicle

Details on study design:

Fifty Sprague Dawley rats/sex (NTac:Sprague Dawley®TMSD®TM) were assigned to two groups of 15 animals/sex (Groups 1 and 4) and two groups of 10 animals/sex (Groups 2 and 3). The animals received either the vehicle control article (corn oil – Group 1) or OSA-thiobiseth formulated in corn oil at dose levels of 100, 300 or 1000 mg/kg/day (Groups 2, 3 and 4, respectively). The test and control articles were administered once daily via oral gavage for 28 consecutive days, at a dose volume of 10.0 mL/kg body weight. Ten animals/sex/dose in Groups 1-4 were sacrificed by CO2 overdose on Day 29 and necropsied. The surviving animals, 5/sex/group in Groups 1 and 4, were sacrificed by CO2 overdose after 14 days of recovery (Day 43) and necropsied.

Examinations

Observations and examinations performed and frequency:

All animals were observed twice daily at least 6 hours apart for moribundity and mortality. Cage side observations were performed daily within 2 hours after the last animal was dosed. On the days of FOB evaluation, including pretest and during recovery, cage side observations were done for each animal scheduled for FOB assessment, prior to removal to the testing room.

Detailed hands-on observations were performed on Day 1 and weekly thereafter (at the time animals were weighed). Signs noted included, but was not limited to, changes in the skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions, and autonomic activity (lacrimation, piloerection, pupil size, and unusual respiratory pattern).

Sacrifice and pathology:

Ten animals/sex/dose, surviving until terminal sacrifice on Day 29 were sacrificed by CO2 overdose and necropsied. The remaining surviving animals, 5/sex in Groups 1 and 4, were sacrificed by CO2 overdose after 14 days of recovery (Day 43) and necropsied. At sacrifice, animals were weighed to the nearest 0.1 gram.

A full necropsy was performed on the terminal and scheduled sacrificed animals on Day 29 (10 animals/sex/dose) and Day 43 (5 animals/sex inGroups 1 and 4), respectively. The scheduled necropsies were performed under the direction and supervision of a pathologist. The necropsy procedure consisted of a thorough and systematic examination and dissection of the animal viscera and carcass, in accordance with BioReliance Standard Operating Procedures. The tissues/organs/gross lesions were collected and fixed in 10% neutral buffered formalin (10% NBF). Gross observations made during necropsy were captured electronically on a computerized, validated Data Capture System (Provantis¿ Version 8.2.0.8).
One Group 2 male (No. 4202) was found dead during acclimation prior to pre-dose FOB observations. This animal was subjected to a full necropsy, per Study Director, but no organ weights or tissues were collected. This animal was replaced by animal No. 4282.

Other examinations:

The following data was evaluated for evidence of toxicity: mortality, clinical signs, body weights and body weight changes, food consumption, clinical pathology, organ weights and macroscopic (gross necropsy) and microscopic pathology. All animals were evaluated in a functional observational battery (FOB) (open field activity, response to stimuli and motor function) prior to the first dose, during the last week of dosing and at the end of the additional 14-day observation period (Groups 1 and 4 only).

Statistics:

The incidence of all effects were calculated by sex and dose. For each sex, body weight, food consumption, grip strength, foot splay, and organ weight data were analyzed by Dunnett's t-test. Clinical pathology was analyzed using ANOVA followed by Dunnett’s t-test. Frequency data such as clinical observations were compared by Fisher's Exact Test, if necessary. All required statistics were determined using either Provantis¿ (Tables and Statistics Version 8.4.0.1), or Minitab® (Version 16.1.0) or Excel (2007).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During post-dose cage side observations, hypersalivation was a prominent test article-related observation in the high dose animals (Group 4, 1000 mg/kg/day) as it was noted in 11/15 males from Days 15 to 28 and 14/15 females from Days 13 to 28. Red-stained nose and/or mouth was also noted in the high dose animals (12/15 males and 6/15 females from Days 23 to 28 and Days 24 to 27, respectively). Labored breathing and red oral or nasal discharge were also noted in 4/15 high dose males.

There were no post-dose cage side observations in the control (Group 1), or low dose (Group 2, 100 mg/kg/day) animals. There was no staining of the nose or mouth and no hypersalivation, or any other observations, noted in the control (Group 1) or in high dose animals (Group 4, 1000 mg/kg/day) during the recovery period. No other observations were noted in the test article treatment groups.

Mortality:

no mortality observed

Description (incidence):

All animals survived until terminal or scheduled sacrifice on Day 29 or Day 43.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose (Group 4, 1000 mg/kg/day) males had a mean body weight that was significantly but slightly (8%) lower than the control; there was no remarkable difference at the end of the recovery period. The lower mean body weight was associated with significantly decreased mean body weight gain for the week 3 and 4 intervals (Days 15 to 22 and Days 22 to 29, respectively) as well as significantly decreased absolute mean body weight gain for the high dose males relative to the control group. The mean % gain in body weight for the high dose males demonstrated a lower gain from Day 1 to 29 (26.75%) relative to the control males (39.94%). No statistically significant differences during the recovery period.

Mean body weights of the treated females were similar to the control throughout the study. However, from Days 15 to 22, the mean body weight gain for the high dose (Group 4, 1000 mg/kg/day) females was statistically significantly lower than control but there was no effect on absolute weight gain from Day 1 through 29, suggesting that this decrease in weight gain may have been incidental and due to normal animal to animal variation. Throughout the remainder of the study, the mean body weight gain was similar between treated and control females.

Food efficiency:

no effects observed

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly higher mean values for neutrophil counts were observed in 300 and 1000 mg/kg/day males and females on Day 29. The mean values for lymphocyte and total leukocyte counts were significantly higher for 100, 300, and 1000 mg/kg/day males, and slightly higher lymphocyte and total leukocyte counts were also noted in 300 and 1000 mg/kg/day females (significant for total leukocyte count in 1000 mg/kg/day females). Monocyte counts were slightly higher in 100 (statistically significant), 300, and 1000 mg/kg/day males. The higher total and differential leukocyte counts in the 1000 mg/kg/day rats may have an association with the nasal cavity inflammatory exudate observed microscopically for this group. The statistically significantly increase in mean eosinophil and basophil was also observed in females treated at 1000 mg/kg/day females.

The statistically lower mean reticulocyte count seen in 1000 mg/kg/day males and the statistically lower mean corpuscular hemoglobin concentration noted in 1000 mg/kg/day females. Based on the small magnitude of the differences, lack of consistent between the sexes and the absence of any related changes there were considered not be be adverse. The remaining hematology data at Day 29 were generally unremarkable and similar among the groups..

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Alterations in the serum chemistry data on Day 29 that were attributed to test article included statistically higher mean values for ALT in 300 and 1000 mg/kg/day males and females and AST in 300 mg/kg/day males and 1000 mg/kg/day males and females. These differences were moderate to marked, generally dose dependent, and consistent with a hepatocellular effect. The higher enzyme activities were accompanied by statistically higher relative liver weights in the 100, 300, and 1000 mg/kg/day females and by centrilobular hypertrophy in the liver of 1000 mg/kg/day males and females. It should be noted that the higher ALT values (>100 U/L) in 100 mg/kg/day male No. 4197 and control female No. 4231 were accompanied by mild hepatic vacuolation.

The statistically higher mean CK values observed in 300 mg/kg/day males and 1000 mg/kg/day males and females were likely an effect of treatment, but there were no clinical or microscopic correlates that would explain the differences.

The slightly, but statistically significantly, higher mean values for sodium in 100, 300, and 1000 mg/kg/day males and chloride in 1000 mg/kg/day males were of no biologic consequence, but may represent mild effects of test article administration. The slightly, but statistically, lower inorganic phosphorus concentrations seen in 300 and 1000 mg/kg/day males on Day 29 were of small magnitude and not dose dependent, but may represent indirect effects of the test article. The lower phosphorus values were not accompanied by similar changes in calcium.

Other statistical differences in the chemistry data on Day 29 were considered incidental to test article administration because of the small magnitude of the difference and/or the lack of a dose response.

At recovery Day 43, the chemistry data were generally unremarkable and comparable between control and 1000 mg/kg/day groups.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The mean urinary osmolality was statistically lower in 1000 mg/kg/day females on Day 29, and was accompanied by slightly lower urine specific gravity (not significant) and higher mean urine volume (not significant). The differences suggest that these females drank more water during the overnight fast; they were not accompanied by meaningful changes in serum urea nitrogen or creatinine in this group and were not attributed to test article administration.

The remaining summary and individual urinalysis data and individual microscopic sediment examination results were generally unremarkable and similar among the groups at Day 29 and between control and 1000 mg/kg/day rats on recovery Day 43.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no remarkable differences in the FOB evaluations (open field observations, elicited response observations, number of urine spots and fecal boli, fore- and hind-limb grip strength, landing foot splay) between the control and treated animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

On Day 29, the increase in absolute and relative weights of adrenal glands in male rats correlated with hypertrophy/hyperplasia of the adrenals noted in the 1000 mg/kg/day male rats. For the remaining alterations in the absolute organ weights (thymus and prostate) and relative organ weights (brain, spleen and testes), there were no correlating microscopic observations. Thus the alterations in these organ weights in the males (thymus, prostate, brain, spleen and testes) were considered to be biologically and toxicologically insignificant. In the liver, centrilobular hypertrophy was noted in 1000 mg/kg/day male rats. However, because this lesion did not persist at 100 mg/kg/day, the increased relative liver weights in 100 mg/kg/day males were also considered to be biologically and toxicologically insignificant.

The increase in the absolute and relative liver weights in 1000 mg/kg/day female rats correlated with the centrilobular hypertrophy noted in that dose group. However, because the centrilobular hypertrophy did not persist in 100 mg/kg/day and 300 mg/kg/day females, the alterations in the relative liver weights in these two groups were considered to be biologically and toxicologically insignificant.

For the remaining other alterations in the absolute organ weights and relative organ weights of both males and females, there were no correlating microscopic observations. Thus, the alterations in these organ weights were considered to be biologically and toxicologically insignificant.

At Day 43, there were several statistically significant differences in the absolute and/or relative organ weights (relative to body weight) in the test article treated animals when compared to the control, however there were no correlating microscopic alterations and thus they were considered to be biologically and toxicologically insignificant.In the absence of other indicators of toxicity, these changes are considered adaptive and generally only rodent-specific.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Description (incidence and severity):

There were no remarkable differences in the FOB evaluations (open field observations, elicited response observations, number of urine spots and fecal boli, fore- and hind-limb grip strength, landing foot splay) between the control and treated animals.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Exudative Inflammation of the Nasal Cavity

the control (Group 1) male rats showed evidence of this lesion, while control female rats did not. This indicates that, at least in the males, the vehicle alone is capable of inducing this lesion, which was exacerbated, based on the incidence and severity, in the high dose animals (Group 4, 1000 mg/kg/day). The lesion was also noted in one low dose (Group 2, 100 mg/kg/day) male with the same severity as the control group. Based on these findings, the No Observed Adverse Effects Level (NOAEL) for this lesion was achieved at 300 mg/kg/day for both sexes.
At Day 43, this lesion of mild intensity persisted in 2/5 1000 mg/kg/day male rats; this lesion was not present for any female rats at Day 43.

Centrilobular Hypertrophy of Liver
Centrilobular hypertrophy was noted in 10/10 male rats and in 8/10 female rats in the high dose group (Group 4, 1000 mg/kg/day). However, this lesion was not noted in the low (Group 2, 100 mg/kg/day) or mid (Group 3, 300 mg/kg/day) dose groups of both sexes; thus, the NOAEL for this lesion was achieved at 300 mg/kg/day for both sexes. This lesion did not persist in the high dose rats of either sex at the end of the recovery period (Day 43).

Hyperplasia of Non-Glandular stomach:
Hyperplasia of the non-glandular stomach was noted in 2/10 male rats and in 4/10 female rats in the high dose groups (Group 4, 1000 mg/kg/day). However, this lesion was not noted in the low or mid dose of both sexes; thus, the NOAEL for this lesion was achieved at 300 mg/kg/day for both sexes. This lesion did not persist in high dose rats of either sex at the end of the recovery period (Day 43).

Hypertrophy/Hyperplasia of Adrenal Cortex
Hypertrophy/hyperplasia of the adrenal cortex was noted in 9/10 high dose (Group 4, 1000 mg/kg/day) male rats, however, this lesion was not noted in the low (Group 2, 100 mg/kg/day) or mid (Group 3, 300 mg/kg/day) dose male rats. Thus, the NOAEL for this lesion was achieved at 300 mg/kg/day in male rats. This lesion did not persist in high dose male rats at Day 43. This lesion was not noted in any female rats at either Day 29 or Day 43.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Target organs for the Substance were noted only in the high dose group (1000 mg/kg/day) and included the nasal cavity, liver, and stomach in males and females and the adrenal glands in the males based upon microscopic observations. Additional observations in the data that may be related to the actions on the target tissues included clinical observations, changes in clinical pathology analytes relative to control and changes in organ and body weights relative to the control, primarily in the males.

Hypersalivation noted post dose, nasal/oral red staining, and nasal/oral discharge were consistent with the observations of mild exudative inflammation of the nasal cavity and clinical pathology changes to the number of leukocyte counts. These findings are interpreted to reflect gavage reflux and possible local irritation by the dosing solution rather than systemic toxicity. The relative absence of these clinical observations in the 1000 mg/kg/day animals after the 14-day recovery period suggests that these effects are transient in nature and do not remain after treatment is halted (Damsch et al, 2011).

Liver centrilobular hypertrophy was associated with statistically higher absolute and/or relative liver weights and moderate to marked increases in ALT and AST, particularly in 1000 mg/kg/day males and females. Again, these changes were not observed in the 1000 mg/kg/day males and females after the 14-day recovery period suggesting that the effects are transient in nature. In the absence of other indicators of toxicity, these changes are considered adaptive and generally only rodent-specific (Hall et al, 2012).

Adrenal hypertrophy in the males, along with the small decrease in weight gain may be associated with the stress accompanying the other changes in these animals. The observation was absent in the recovery animals, which again suggests the transient nature of the change.

REFERENCES
Damsch, S., Eichenbaum, G., Tonelli, A., Lammens, L., Van den Bluck, K., Feyen, B., Vandenberghe, J., Megens, A., Knight, E., & Kelly, M. (2011). Gavage-related reflux in rats : Identification, pathogenesis, and toxicological implications (review). Toxicol Pathol, 39(2), 348-360. doi: 10.1177/0192623310388431

Hall, A.P., Elcombe, C.R., Foster, J.R., Harada,T., Kaufmann,W., Knippel, A., Küttler, K., Malarkey D.E., Maronpot, R.R., Nishikawa, A.,Nolte, T., Schulte, A., Strauss, V., York, M.J. (Oct. 2012). Toxicologic Pathology. Toxicol Pathol, Vol. 40 No. 7, 971-994. doi: 10.1177/0192623312448935

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Organ:

adrenal glands

nasal cavity

stomach

Treatment related:

yes

Dose response relationship:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, the Substance given to male and female Sprague Dawly rats by oral gavage, once daily in corn oil for 28 days was associated with no mortality, no effects on food consumption and no remarkable observations in a functional observational battery at doses up to 1000 mg/kg/day. The no-observable-adverse effect level (NOAEL) was 300 mg/kg/day based upon an absence of remarkable clinical signs or significant body weight alterations, and a lack of microscopic correlates for the clinical pathology alterations and organ weight changes. Target organs for the Substance noted at 1000 mg/kg/day included the nasal cavity, liver, and stomach in males and females and the adrenal glands in the males.

Only mild exudative inflammation of the nasal cavity persisted in 2 of 5 1000 mg/kg/day males after a 14-day recovery period. There were no other remarkable observations in the 1000 mg/kg/day group during the recovery period, indicating that the effects noted during the 28-day treatment period did not persist and did not result in any additional delayed effects after a 14-day recovery period.

Executive summary:

This study assessed the toxicity following repeated oral administration (gavage) in Sprague Dawley rats and evaluated the reversibility, persistence or onset of effects after 14 days post treatment.

Fifty Sprague Dawley rats/sex (NTac:Sprague Dawley^®TMSD^®TM) were assigned to two groups of 15 animals/sex (Groups 1 and 4) and two groups of 10 animals/sex (Groups 2 and 3). The animals received either the vehicle control article (corn oil – Group 1) or the Susbtance formulated in corn oil at dose levels of 100, 300 or 1000 mg/kg/day (Groups 2, 3 and 4, respectively). The test and control articles were administered once daily via oral gavage for 28 consecutive days, at a dose volume of 10.0 mL/kg body weight. Ten animals/sex/dose in Groups 1-4 were sacrificed by CO2overdose on Day 29 and necropsied. The surviving animals, 5/sex/group in Groups 1 and 4, were sacrificed by CO2overdose after 14 days of recovery (Day 43) and necropsied.

The following data was evaluated for evidence of toxicity: mortality, clinical signs, body weights and body weight changes, food consumption, clinical pathology, organ weights and macroscopic (gross necropsy) and microscopic pathology. All animals were evaluated in a functional observational battery (FOB) (open field activity, response to stimuli and motor function) prior to the first dose, during the last week of dosing and at the end of the additional 14-day observation period (Groups 1 and 4 only).

The Substance had no effect on mortality or food consumption. There were generally no remarkable test article-related differences in the remainder of the data between animals receiving Substance at 300 mg/kg/day or less relative to the control group and generally no differences between the high dose (1000 mg/kg/day) animals and the control after the completion of the 14-day recovery period.

Hypersalivation noted post dose beginning on Day 15 and Day 13 in the 1000 mg/kg/day males and females, respectively, was a prominent treatment-related observation. This was accompanied by red staining of the nose and/or mouth, labored breathing, and/or red oral/nasal discharge in this group near the end of the treatment period. These clinical observations were consistent with the stated microscopic changes noted in the nasal passages. Yellow staining of the urogenital area was also noted in the 1000 mg/kg/day group. There were no remarkable differences in clinical signs between the control and high dose animals during the recovery period.

There were no remarkable differences in the FOB evaluations (open field observations, elicited response observations, number of urine spots and fecal boli, fore- and hind-limb grip strength, landing foot splay) between the control and treated animals.

The Substance had no effect on body weight or body weight gain in females, or in males receiving 300 mg/kg/day or less. On Day 29, males receiving 1000 mg/kg/day had slightly (8%) but statistically significantly lower mean body weight than the control males. Mean body weight gain of these males was significantly lower than control from Days 15 to 22 and again from Days 22 to 29. Mean body weight and mean body weight gain of the 1000 mg/kg/day males and females was similar to the control during the recovery period.

On Day 29, changes in the hematology data included significantly higher mean values for neutrophil counts in 300 and 1000 mg/kg/day males and females and for lymphocyte and total leukocyte counts in 100, 300, and 1000 mg/kg/day males (was also significant for total leukocyte count in 1000 mg/kg/day females). These observations may represent indirect effects of treatment as a result of associated nasal cavity inflammation and clinical signs of red staining and/or discharge in the nasal and oral areas. Serum chemistry alterations on Day 29 that were attributed to treatment included statistically higher mean values for alanine aminotransferase (ALT) in 300 and 1000 mg/kg/day males and females and aspartate aminotransferase (AST) in 300 mg/kg/day males and 1000 mg/kg/day males and females. The increased ALT and AST values were associated with the statistically significant increases in liver weight and centrilobular hypertrophy in animals given 1000 mg/kg/day. Statistically significantly higher mean creatine kinase (CK) values observed in 300 mg/kg/day males and 1000 mg/kg/day males and females were also likely effects of the test article but there were no other clinical or microscopic correlates to explain these differences.

The urinalysis, urine osmolality and coagulation data on Day 29 were unremarkable and similar between the treated animals and the control animals. On recovery Day 43, the clinical pathology data were generally unremarkable and comparable between control and 1000 mg/kg/day animals.

The Substance was associated with increased absolute and relative adrenal gland weights in 1000 mg/kg/day male rats that correlated with hypertrophy/hyperplasia of the adrenals noted in this group. The increase in the absolute and relative liver weights in 1000 mg/kg/day female rats correlated with the hepatic centrilobular hypertrophy noted in this group. Target organs for both sexes given the Substance at 1000 mg/kg/day, based upon microscopic examination, included the nasal cavity (exudative inflammation), liver (centrilobular hypertrophy) and stomach (hyperplasia of the nonglandular portion), and in the male rats only, the adrenal glands (hypertrophy/hyperplasia of the cortex). Generally, none of these microscopic lesions were noted in males or females given the susbtance at 300 mg/kg/day or less, or in the 1000 mg/kg/day animals at the end of the 14-day recovery period, indicating that these changes did not persist without treatment.

In conclusion, treatment to the male and female Sprague Dawly rats by oral gavage, once daily in corn oil for 28 days was associated with no mortality, no effects on food consumption and no remarkable observations in a functional observational battery at doses up to 1000 mg/kg/day. The no-observable-adverse effect level (NOAEL) was 300 mg/kg/day based upon an absence of remarkable clinical signs or significant body weight alterations, and a lack of microscopic correlates for the clinical pathology alterations and organ weight changes. Target organs for the Substance noted at 1000 mg/kg/day included the nasal cavity, liver, and stomach in males and females and the adrenal glands in the males.

Only mild exudative inflammation of the nasal cavity persisted in 2 of 5 1000 mg/kg/day males after a 14-day recovery period. There were no other remarkable observations in the 1000 mg/kg/day group during the recovery period, indicating that the effects noted during the 28-day treatment period did not persist and did not result in any additional delayed effects after a 14-day recovery period.