Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 September 2015 through 7 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study is required under Annex IX. There is no existing data that is close enough to use for read-across. Further, there are not models that can adequately predict the effects seen in a 90-day repeat dose study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyronitrile
EC Number:
203-700-6
EC Name:
Butyronitrile
Cas Number:
109-74-0
Molecular formula:
C4H7N
IUPAC Name:
butanenitrile
Details on test material:
Sponsor's Identification: 98-0254,
ACC No.: 900741,
Lot/Batch No.: T-11
Date Received: September 21, 1999
Physical Description: Transparent, colorless liquid
Storage Conditions: Room temperature
Specific details on test material used for the study:
Identification: n-Butyronitrile, Lot no. TXTXOL [ID no. 150165]
Physical description: Clear liquid
Date of receipt: 12-May-2015

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Crl:CD(SD) rats were used as the test system for this study. This species and strain of animal is recognized as appropriate for subchronic toxicity studies. The Sprague Dawley rat was selected because it is a widely used strain for which significant historical control data are available. The number of animals selected was based on regulatory guidelines (OECD Test Guideline 408 “Repeated Dose 90-Day Oral Toxicity in Rodents”). A minimum of 10 animals/sex/group was necessary for repeated-dose rodent studies in order to provide information on the possible health hazards likely to arise from prolonged exposure. These recommendations helped to ensure that the number of animals that survive until the end of the study will be sufficient to permit a meaningful evaluation of toxicological effects.

Crl:CD(SD) rats (45 males and 45 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 22-Sep-2015. The animals were approximately 35 days old at receipt. Each animal was examined by a qualified technician on t he day of receipt and weighed on the following day. Each animal was uniquely identified with a subcutaneous microchip (BMDS system) implanted in the dorsoscapular area. All animals were housed for a 16-day acclimation. During this period, each animal was observed twice daily for mortality and changes in general appearance or behavior. Data collection during acclimation began on 23-Sep-2015. Individual body weights and cage food weights were recorded and detailed physical examinations were performed periodically during acclimation. Ophthalmic examinations were performed on all animals during acclimation prior to randomization.

Upon arrival, all animals were housed 2 to 3 per cage by sex in clean, solid bottom cages containing ground corncob bedding material (Bed O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). Following placement into treatment groups, animals were housed in groups of 2 of the same sex per cage. The cages were cleaned and changed routinely at a frequency consistent with maintaining good animal health. The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. The animals were temporarily separated as necessary to allow for the performance of protocol-specified activities. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at Charles River are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), is a certified feed with appropriate analyses performed by the manufacturer and provided to Charles River. Reverse osmosis-treated (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the study, except during the period of fasting prior to clinical pathology blood collection and during the conduct of FOBs and motor activity assessments when food, but not water, was withheld. Municipal water supplying the facility was analyzed for contaminants according to SOPs. The results of the diet and water analyses are maintained at Charles River. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. All animals were housed throughout acclimation and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 ± 5°F (22 ± 3°C) and 50 ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in Appendix F. Actual mean daily temperature ranged from 69.8°F to 70.7°F (21.0°C to 21.5°C) and mean daily relative humidity ranged from 40.2% to 48.2% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. The 12-hour light/12-hour dark photoperiod was interrupted as necessary to allow for the performance of protocol-specified activities. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
The vehicle and test substance formulations were administered orally by gavage via an appropriately sized flexible, disposable, plastic ball-tipped dosing cannula (Instech Solomon, Plymouth Meeting, PA) once daily for 90 or 91 consecutive days, through the day prior to the scheduled necropsy. The dose volume for all groups was 10 mL/kg. Individual doses were based on the most recently recorded body weights to provide the correct mg/ kg/day dosage. Adjusted doses became effective the day of collection of the weekly body weights. The first day of dosing was study day 0; the first week of dosing was study week 0.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
During the validation study, test substance homogeneity and stability following at least 10 days of room temperature and refrigerated storage were established for test substance concentrations in the vehicle at 1 and 50 mg/mL. Therefore, stability assessment was only conducted on the lowest concentration of 0.7 mg/mL in this study. Concentration, homogeneity and resuspension homogeneity of the dosing formulations was conducted on the first formulation preparation used for dosing on the study. Test substance formulations of sufficient volume for dosing a group of animals for up to 1 week (length and volume dependent on established stability) were prepared. Four 1.0-mL samples were collected from the top, middle, and bottom of the lowest and highest concentrations of test substance formulations and from the middle of the control and mid-dose formulations. Two samples from each stratum were analyzed to assess the concentration and/or homogeneity of the test substance in the mixtures. The remaining samples were stored at room temperature (18°C to 24°C) as backup samples. The samples from the lowest concentration (0.7 mg/mL) were also used as time-zero data for comparison to results obtained from later stability samples. To establish resuspension homogeneity and stability at the lowest concentration (0.7 mg/mL), a volume of formulation similar in size to the amount needed for 1 day from the low and high dose formulations was stored at room temperature (18°C to 24°C) for at least 10 days. After remixing for a minimum of 30 minutes using a magnetic stirrer, four 1.0-mL samples were collected from the top and bottom of the formulations. Two samples from each stratum were analyzed to assess homogeneity after storage and resuspension. Stability of the lowest concentration (0.7 mg/mL) was evaluated comparing the inter-strata means to the overall concentration from the bulk homogeneity data (time-zero). The remaining samples were stored at room temperature (18°C to 24°C) as backup samples. Concentration was also assessed on the formulations prepared for dosing during study weeks 3, 7, and 12. Four 1.0-mL samples were collected from the middle of the control and each test substance formulation. Two samples from each strata were analyzed to assess the concentration of the test substance in the mixtures; the remaining samples were stored at room temperature (18°C to 24°C) as backup samples. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated gas chromatography (GC) method using flame ionization (FID) detection.
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
7 mg/kg bw (total dose)
Remarks:
Males
Dose / conc.:
15 mg/kg bw (total dose)
Remarks:
Males
Dose / conc.:
30 mg/kg bw (total dose)
Remarks:
males
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
Females
Dose / conc.:
25 mg/kg bw (total dose)
Remarks:
Females
Dose / conc.:
50 mg/kg bw (total dose)
Remarks:
Females
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Standard OECD 408 study design, see additonal Attachment of study design below
Positive control:
no

Examinations

Observations and examinations performed and frequency:
Clinical examinations were performed at the time of dose administration and 1-2 hours following dose administration. The absence or presence of findings was recorded for individual animals at the scheduled intervals. Detailed physical examinations were conducted on all animals within 4 days of receipt, on the day of randomization, weekly (± 2 days) during the study period, and on the days of the scheduled necropsy. Due to social housing, some observations could not be attributed to a single animal.
Sacrifice and pathology:
A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Clinical findings that were confirmed macroscopically were designated CEO on the individual macroscopic data tables. The following tissues and organs were collected and placed in 10% neutral-buffered formalin (except as noted):

Adrenals (2) Aorta
Bone with marrow
Femur
Sternum
Bone marrow smear (from femur)a
Brainb Cervix
Epididymides(2)c Eyes with optic nerve(2)d
Gastrointestinal tract Esophagus
Stomach Duodenum
Jejunum Ileum
Cecum Colon
Rectum Heart
Kidneys(2) Larynx
Liver (sections of 2 lobes) Pancreas
Lungs (including bronchi, fixed by inflation with fixative)
Lymph nodes Ovaries (2) with oviductse
Axillary (2) Peripheral nerve (sciatic)
Mandibular (2) Peyer’s patches
Mesenteric Pharynx
Pituitary Prostate
Salivary glands (mandibular [2]) Seminal vesicles (2)
Skeletal muscle (rectus femoris) Skin with mammary gland f
Spinal cord (cervical, thoracic, lumbar) Spleen
Testes (2)c Thymus
Thyroid (with parathyroids [2])e Tongue
Trachea Urinary bladder
Uterus Vagina
Gross lesions (when possible)
a = Bone marrow smears were obtained at scheduled necropsy, but not placed in formalin; slides were not examined.
b = Histologic trimming of the brain was performed per Charles River SOP such that 7 cross (coronal) sections of the brain were taken.
c = Fixed in modified Davidson’s solution
d = Fixed in Davidson’s solution
e = Oviducts and parathyroids were examined if in plane of section and in all cases where a gross lesion of the organ was present.
f = For females; a corresponding section of skin was taken from the same anatomic area in males.

Statistics:
Please see "Any other information on materials and methods incl. tables" for details as the character limit in this box prevents the full description of the statistical methods.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related clinical observations from study week 1 until the end of the study included wet clear material on various body surfaces (mouth, neck, trunk, and forelimbs) and salivation. These observations were present in the 15 and 30 mg/kg/day group males and 25 and/or 50 mg/kg/day group females in a dose responsive manner, and at a very limited incidence in the 7 mg/kg/day group males and10 mg/kg/day group females. All other clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner, and/or were common findings for laboratory rats of this age and strain
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Female no. 5711 (control group) was euthanized in extremis on study day 85 due to injuries that occurred from a mechanical trauma (accidently shut in the cage). All other animals survived to the scheduled necropsy
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related lower mean body weights were observed in the 30 mg/kg/day group males at study week 3 and continuing throughout the remainder of the study. These differences were not statistically significant and were no more than 7.5% lower throughout the dosing period. Correlating lower mean body weight gains were generally observed in the 30 mg/kg/day males, but were statistically significant only during study weeks 3 to 4 and 6 to 7. The cumulative body weight gain of the 30 mg/kg/day group at the end of the dosing period (study week 0 to 13) was 12.4% less than that of the concurrent control group.

A statistically significant higher body weight gain was observed in the 50 mg/kg/day group females during study weeks 1 to 2, resulting in a higher mean body weight. Although subsequent body weight gains in the 50 mg/kg/day group females were generally consistent with the control group, the mean body weight for the 50 mg/kg/day group females remained higher throughout the study.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related hematologic alterations were noted in the 30 mg/kg/day group males and 50 mg/kg/day group females. There were no test substance-related changes in coagulation parameters.

Statistically significant test substance-related higher mean reticulocyte (percent and absolute) values and lower mean red cell hemoglobin concentration (MCHC) values were observed in the 30 mg/kg/day group males and the 50 mg/kg/day group females, and higher mean red cell volume (MCV) were observed in the 50 mg/kg/day group females. These changes were consistent with a regenerative hematologic response.

There were no other test substance related effects on hematology parameters. However, some statistically significant differences were observed when the control and test substance treated groups were compared. Higher absolute white blood cell, large unstained cell, lymphocyte, and monocyte values were noted in the 15 mg/kg/day group males and lower mean cell hemoglobin (MCH) was noted in the 10 mg/kg/day group females. All group mean values were within the Charles River historical control range and a dose related response was not observed; therefore, these changes were considered due to biologic variation and not administration of the test substance.

Lower mean red cell distribution width (RDW) values were observed in the 15 and 30 mg/kg/day group males and the 50 mg/kg/day group females and lower mean hemoglobin distribution width (HDW) values were observed in the 15 and 30 mg/kg/day group males and the 25 and 50 mg/kg/day group females. These changes were in a direction of no known toxicologic significance, and as such were considered unrelated to the test substance.

Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related serum chemistry alterations were noted in the 7, 15, and 30 mg/kg/day group males and 25 and 50 mg/kg/day group females.

Statistically significant higher mean total bilirubin (TBIL) values were noted in the 30 mg/kg/day group males and 50 mg/kg/day group females, and st atistically significant higher mean aspartate aminotransferase (AST) values were noted in the 30 mg/kg/day group males. In the absence of other changes in hepatic serum chemistry parameters, and in the presence of perturbations in several hematologic parameters, these serum chemistry changes are suggestive of damage to erythrocytes (hemolysis) and considered adverse.

Statistically significant lower mean cholesterol values were noted in the 7, 15, and 30 mg/kg/day group males and 25 and 50 mg/kg/day group females. Although, this chang e is considered test-substance related, it is likely toxicologically irrelevant.

There were no other test substance-related effects on serum chemistry parameters. However, some statistically significant differences were observed when the control and test substance treated groups were compared. Higher mean albumin value was noted in the 15 mg/kg/day group males. As a dose-related response was not observed, this change was considered spurious and not related to administration of the test substance.
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related higher heart, liver, and spleen weights were noted in the 25 and/or 50 mg/kg/day group females.

Statistically significant higher mean heart weights (absolute and relative to brain weights) were noted in the 50 mg/kg/day group females. A histologic correlate was not observed.

Statistically significant higher mean liver weights (absolute and relative to body and brain weights) were noted in the 50 mg/kg/day group females. Statistically significant higher mean liver weights (relative to body weight) were observed at 25 mg/kg/day. Although not statistically significant, higher mean liver weights (absolute and relative to brain weight) which were outside Charles River historical control mean ± 2 SD, were noted in the 25 mg/kg/day gr oup females. The liver weight change at 50 mg/kg/day possibly correlated with the histopathologic observation of cytoplasmic rarefaction in the 50 mg/kg/day group females.

Statistically significant higher mean spleen weights (absolute and relative to body and brain weights) were noted in the 50 mg/kg/day group females. A histologic correlate was not observed.

In the males, some organ weight differences were statistically significant when compared t o the control group but were considered to be a result of a test substance -related effect on final body weight. These included higher adrenal gland weights relative to body weight, heart weights relative to body weight, liver weights relative to body weight, and spleen weights relative to body weight in the 30 mg/kg/day group.

There were no other test substance-related effects on organ weights. However, some statistically significant differences were observed when the control and test substance treated groups were compared. Lower testes weights (absolute and relative to brain weights) were noted in the 30 mg/kg/day group males. This change was due mainly to a low individual testes weight in 1 male (no. 5657). All group means were within Charles River historical control mean ± 2 SD; therefore, this change was considered due to biologic variation and not the test substance.

Higher ovaries/oviduct weights (absolute and relative to body and brain weights) were noted in the 50 mg/kg/day group females. A histologic correlate was not observed. Additionally, all group means were within Charles River historical control mean ± 2 SD; therefore, this change was considered due to biologic variation and not the test substance.

Higher relative (to body and brain weight) liver weights and lower absolute seminal vesicle/prostate weights were observed in the 15 mg/kg/day group males. These changes lacked a dose-related response and, therefore, were considered due to biologic variation and not administration of the test substance.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related microscopic findings were noted in the liver of the 7, 15, and 30 mg/kg/day group males and the 25 and 50 mg/kg/day group females and the spleen of the 7, 15, 30 mg/kg/day group males.

Test substance-related accumulation of pigment was noted in the spleen of the 7, 15, and 30 mg/kg/day group males. The pigment was light tan to golden brown and within the cytoplasm of macrophages in the red pulp sinuses. This accumulation of pigment within the spleen is most likely hemosiderin deposition, which results when splenic macrophages engulf and degrade hemoglobin following red blood cell (hemolysis). This finding was also noted in the 50 mg/kg/day group females; however, a comparable number of spleens from the control group females also contained similar brown pigment. Therefore, in the 50 mg/kg/day group females, the brown pigment in the spleen was considered not related to administration of the test substance.

Test substance-related hepatocellular cytoplasmic rarefaction (glycogen, presumptive) was noted in the liver of the 7, 15, and 30 mg/kg/day group males and 25 and 50 mg/kg/day group females. This change was characterized by wispy vacuolation of hepatocytes, and was mainly observed in the periportal to midzonal regions, but was panlobular in some animals. In the females this change possibly correlated with increased liver weights. The hepatocellular cytoplasmic rarefaction was not associated with changes in liver enzymes, evidence of hepatocellular degeneration, or decrease in liver function.

Hepatocellular vacuolation characterized by accumulation of round, discrete macrovesicular and microvesicular vacuoles (lipid, presumptive) was also observed. This change was similar in incidence and severity when the control and test substance-treated groups were compared and was considered likely due to the corn oil vehicle.

There were no other test substance-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw (total dose)
Sex:
male
Basis for effect level:
clinical biochemistry
gross pathology
haematology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw (total dose)
Sex:
female
Basis for effect level:
clinical biochemistry
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

The Tables S1 - S6, S29 - S32, S39, S42 and S43 show the summary data

Table SI (Males)

Project No.:WIL-387065M

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.      

                             Summary of Survival And Disposition

Males

Group:

1

2

3

4

Week

live

FD

EE

SE

live

FD

EE

SE

live

FD

EE

SE

live

FD

EE

SE

0

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

1

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

2

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

3

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

4

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

5

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

6

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

7

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

8

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

9

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

10

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

11

10

0

0

0

10

0

0

0

10

0

0

0

10

0

0

0

12

5

0

0

5

5

0

0

5

5

0

0

5

5

0

0

5

13

0

0

0

5

0

0

0

5

0

0

0

5

0

0

0

5

Week = Week of Study

FD = Found Dead

EE = Euthanized in Extremis

SE = Scheduled Euthanasia

1-    0 mg/kg/Day

2-  7 mg/kg/Day

3-  15 mg/kg/Day

4-  30 mg/kg/Day

Table S2 (Females)

Project No.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor:EASTMAN CHEMICAL CO. 

                                  Summary of Survival And Disposition

Females

Group:

1

2

3

4

Week

live

FD

AD

EE

SE

live

FD

AD

EE

SE

live

FD

AD

EE

SE

live

FD

AD

EE

SE

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

1

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

2

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

3

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

4

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

5

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

6

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

7

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

8

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

9

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

11

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

10

0

0

0

0

12

4

0

1

0

5

5

0

0

0

5

5

0

0

0

5

5

0

0

0

5

13

0

0

0

0

4

0

0

0

0

5

0

0

0

0

5

0

0

0

0

5

Week = Week of Study

FD = Found Dead

AD = Accidental Death

EE = Euthanized in Extremis

SE = Scheduled Euthanasia

1 - 0 mg/kg/Day

2 - 10 mg/kg/Day

3 - 25 mg/kg/Day

4 - 50 mg/kg/Day

Table S3  (Detailed Physical Examinations/Dispositions - Males)

Project no.:WIL-387065M

A 90-Day Oral Study of N-Butyronitrile in Rats                                             

Sponsor: EASTMAN CHEMICAL CO.

Summrary of Clinical Findings:   Total Occurrence/no. of Animals

Male

Table Range:

Day 000 TO Day 091

Group:

1

2

3

4

normal

-no significant Clinical Observations

126/10

130/10

130/10

118/10

Disposition

-Scheduled Euthanasia; Primary (Day 90/91)

10/10

10/10

10/10

10/10

Body/Integument

 -Hair Loss Facial Area

4/ 1

0/ 0

0/ 0

5/ 2

Eyes/Ears/nose

-dried red material around nose

0/ 0

0/ 0

0/ 0

1/ 1

Body/Integument

-Scabbing Facial Area

2/ 1

0/ 0

0/ 0

8/ 3

Body/Integument

-wet Yellow material Urogenital Area

0/ 0

0/ 0

0/ 0

2/ 2

1 -  0 mg/kg/Day 

2 -  7 mg/kg/Day

3 -  15 mg/kg/Day

4 -  30 mg/kg/Day

Table S4 (Detailed Physical Examinations/Dispositions - Females)

Project no.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats                                           

Sponsor: EASTMAN CHEMICAL CO.

Summary of Clinical Findings:   Total Occurrence/no. of Animals

Female

Table Range:

Day 000 To Day 091

Group:

1

2

3

4

normal

-no significant Clinical Observations

128/10

125/10

130/10

127/10

Disposition

-Accidental Death

1/ 1

 0/ 0

 0/ 0

 0/ 0

 -Scheduled Euthanasia; Primary (Day 90/91)

9/ 9

 10/10

 10/10

 10/10

Behaviour/CNS

-Hypoactivity

1/ 1

 0/ 0

 0/ 0

 0/ 0

Body/Integument

-Hair Loss Forelimb(S)

 0/ 0

4/ 1

 0/ 0

2/ 1

-Hair Loss dorsal neck

 0/ 0

1/ 1

 0/ 0

 0/ 0

Cardio-Pulmonary

-Pale Extremities

1/ 1

 0/ 0

 0/ 0

 0/ 0

-Cool Body

1/ 1

 0/ 0

 0/ 0

 0/ 0

-Laboured Respiration

1/ 1

 0/ 0

 0/ 0

 0/ 0

 -Rales

1/ 1

 0/ 0

 0/ 0

 0/ 0

 -Gasping

1/ 1

 0/ 0

 0/ 0

 0/ 0

Eyes/Ears/nose

 -Pupil Dilated Left Eye

1/ 1

 0/ 0

 0/ 0

 0/ 0

Eyes/Ears/nose

-Pupil Dilated Right Eye

1/1

0/ 0

0/ 0

0/ 0

-dried red material around nose

1/ 1

0/ 0

0/ 0

1/ 1

-Partial Closure Left Eye

1/ 1

0/ 0

0/ 0

0/ 0

-Partial Closure Right Eye

1/ 1

0/ 0

0/ 0

0/ 0

-dried red material Facial Area

1/ 1

0/ 0

0/ 0

0/ 0

Body/Integument. II

-Scabbing Facial Area

1/ 1

0/ 0

0/ 0

0/ 0

1 -  0 mg/kg/Day 

2 -  7 mg/kg/Day

3 -  15 mg/kg/Day

4 -  30 mg/kg/Day

Table S5 (Dosing Day Observations - Males)

Project no.:WIL-387065M

90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

 Summary of Post-Dose Findings: Total Occurrence/no. of Animals

Male

Table Range:

Day 0 To Day 90

Group:

1

2

3

4

normal

Time of Dose

-no significant Clinical Observations

902/10

902/10

902/10

874/10

1-2 hours Post Dosing

-no significant Clinical Observations

892/10

893/10

708/10

307/10

Unscheduled Observations (>120 Mins)

-no significant Clinical Observations

 7/ 7

 0/0

 0/0

0/ 0

Eyes/Ears/nose

Time of Dose

-dried red material around nose

3/2

2/2

3/2

3/3

1-2 hours Post Dosing

-dried red material around nose

 5 /4

3/3

2/1

0/ 0

Body/Integument

Time of Dose

-wet Clear material Forelimb(S)

0/ 0

0/ 0

0/ 0

1/1

1-2 hours Post Dosing

-wet Clear material Forelimb(S)

0/ 0

G/G

2/2

 15/8

-wet Clear material VENTRAL neck

0/ 0

G/G

1/1

 36/7

Oral/Dental

Time of Dose

-wet Clear material around mouth

0/ 0

0/ 0

1/1

 20/5

-Salivation

0/ 0

0/ 0

0/ 0

12/2

1-2 hours Post Dosing

-wet Clear material around mouth

0/0

9/4

167/10

557/10

-wet Yellow material around mouth

0/0

0/0

4/3

18/8

-dried Yellow material around mouth

0/0

0/0

6/5

5/4

-wet red material around mouth

0/0

0/0

0/0

1/1

-dried red material around mouth

0/0

0/0

5/3

0/0

-Salivation

1/1

1/1

19/6

49/10

-Erosion of lower Lip

0/0

0/0

1/1

0/0

Special

Time of Dose

 -reddened Left ear

0/0

1/1

0/0

0/0

 -reddened Right ear

0/0

1/1

0/0

0/0

Body/Integument III

1-2 hours Post Dosing

-wet Yellow material Urogenital Area

0/0

0/0

1/1

0/0

 -wet Clear material dorsal Trunk

0/0

0/0

0/0

2/1

Special II

Time of Dose

 -undetermined Amount of Dose Expelled

0/0

1/1

0/0

1/1

1 -  0 mg/kg/Day 

2 -  7 mg/kg/Day

3 -  15 mg/kg/Day

4 -  30 mg/kg/Day

Table S6  (Dosing Day Observations - Females)

Project no.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor:EASTMAN CHEMICAL CO.

Summary of Post-Dose Findings: Total Occurrence/no. of Animals

Female

Table Range:

Day 0 To Day 90

Group:

1

2

3

4

normal

Time of Dose

-no significant Clinical Observations

897/10

903/10

899/10

893/10

1-2 hours Post Dosing

-no significant Clinical Observations

899/10

900/10

827/10

583/10

Behaviour/CNS

Time of Dose

 -Excessive Struggling During Dosing

0/0

0/0

0/0

1/1

Cardio-Pulmonary

1-2 hours Post Dosing

-Laboured Respiration

1/1

0/0

0/0

0/0

 -Gasping

1/1

0/0

0/0

0/0

Eyes/Ears/nose

Time of Dose

-dried red material around nose

3/3

2/1

2/1

4/2

1-2 hours Post Dosing

 -Clear Discharge Left Eye

0/0

0/0

0/0

1/1

-Pupil Dilated Left Eye

1/1

0/0

0/0

0/0

-Pupil Dilated Right Eye

1/1

0/0

0/0

0/0

-dried red material around nose

1/1

1/1

3/2

6/4

Body/inTEG. II

1-2 hours Post Dosing

 -wet Clear material Forelimb(s)

0/0

0/0

0/0

2/2

-wet Clear material ventral neck

0/0

1/1

0/0

6/3

-wet Clear material ventral trunk

0/0

0/0

0/0

1/1

Oral/Dental

Time of Dose

-wet Clear material around mouth

0/0

0/0

3/3

4/3

-Salivation

0/0

0/0

1/1

3/2

1-2 hours Post Dosing

-wet Clear material around mouth

0/0

3/2

57/10

284/10

-wet Yellow material around mouth

0/0

0/0

1/1

4/4

-dried Yellow material around mouth

0/0

0/0

6/5

7/5

-wet red material around mouth

0/0

0/0

1/1

3/2

-dried red material around mouth

1/1

0/0

6/3

5/4

-Salivation

0/0

0/0

6/5

20/9

Body/Integument III

1-2 hours Post Dosing

-wet Yellow material Urogenital Area

0/0

0/0

0/0

2/2

Special II

Time of Dose

-Undetermined Amount of Dose Expelled

2/2

0/0

0/0

0/0

1 -  0 mg/kg/Day 

2 -  7 mg/kg/Day

3 -  15 mg/kg/Day

4 -  30 mg/kg/Day

Table S29 (Males)

Project no.:WIL-387065M

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of Hematology And Coagulation Values

Males

Analysis Group:

0 mg/kg/Day

7 mg/kg/Day

15 mg/kg/Day

30 mg/kg/Day

WBC (thous/µL)

Mean

8.43

9.37

11.65**

9.21

% Difference

11.2

38.2

9.3

S.D.

1.196

1.169

2.302

1.843

S.E.

0.399

0.370

0.728

0.614

N

9

10

10

9

RBC (mil/µL)

Mean

9.40

9.57

9.72

8.83

% Difference

1.8

3.4

-6.1

S.D.

0.557

0.499

0.460

0.484

S.E.

0.186

0.158

0.145

0.161

N

9

10

10

9

HGB (g/dL)

Mean

16.2

16.4

16.6

15.3

% Difference

1.2

2.5

-5.6

S.D.

0.73

0.61

0.87

0.76

S.E.

0.24

0.19

0.28

0.25

N

9

10

10

9

HCT (%)

Mean

49.5

50.3

51.7

48.3

% Difference

1.6

4.4

-2.4

S.D.

1.91

3.17

3.11

1.97

S.E.

0.64

1.00

0.98

0.66

N

9

10

10

9

MCV (fL)

Mean

52.8

52.6

53.2

54.8

% Difference

-0.4

0.8

3.8

S.D.

1.97

1.93

1.23

2.38

S.E.

0.66

0.61

0.39

0.79

N

9

10

10

9

MCH (pg)

Mean

17.2

17.2

17.1

17.4

% Difference

0.0

-0.6

1.2

S.D.

0.60

0.50

0.32

0.69

S.E.

0.20

0.16

0.10

0.23

N

9

10

10

9

MCHC (g/dL)

Mean

32.7

32.7

32.1

31.7*

% Difference

0.0

-1.8

-3.1

S.D.

0.30

0.87

0.65

0.68

S.E.

0.10

0.28

0.21

0.23

N

9

10

10

9

PLATELET (thous/µL)

Mean

950.

988.

926.

844.

% Difference

4.0

-2.5

-11.2

S.D.

222.4

194.2

115.7

129.9

S.E.

74.1

61.4

36.6

43.3

N

9

10

10

9

PT (seconds)

Mean

16.5

16.2

16.5

16.7

% Difference

-1.8

0.0

1.2

S.D.

0.93

0.63

0.64

0.87

S.E.

0.29

0.20

0.20

0.27

N

10

10

10

10

APTT (seconds)

Mean

14.7

14.1

13.9

14.0

% Difference

-4.1

-5.4

-4.8

S.D.

2.98

2.36

1.94

2.68

S.E.

0.94

0.75

0.61

0.85

N

10

10

10

10

RETIC (%)

Mean

1.6

1.5

1.7

2.4**

% Difference

-6.3

6.3

50.0

S.D.

0.24

0.11

0.20

0.72

S.E.

0.08

0.03

0.06

0.24

N

9

10

10

9

RETIC ABSOLUTE (thous/µL)

Mean

147.1

139.5

159.2

209.4**

% Difference

-5.2

8.2

42.4

S.D.

21.12

12.95

23.01

52.47

S.E.

7.04

4.09

7.28

17.49

N

9

10

10

9

MPV (fL)

Mean

7.41

7.15

7.25

7.66

% Difference

-3.5

-2.2

3.4

S.D.

0.647

0.679

0.932

1.015

S.E.

0.216

0.215

0.295

0.338

N

9

10

10

9

NEU (%)

Mean

17.0

16.3

15.1

20.2

% Difference

-4.1

-11.2

18.8

S.D.

3.47

4.27

4.57

4.80

S.E.

1.16

1.35

1.45

1.60

N

9

10

10

9

LYMPH (%)

Mean

78.4

79.9

80.6

75.2

% Difference

1.9

2.8

-4.1

S.D.

3.20

4.61

4.98

5.15

S.E.

1.07

1.46

1.58

1.72

N

9

10

10

9

MONO (%)

Mean

2.1

2.0

2.4

2.4

% Difference

-4.8

14.3

14.3

S.D.

0.47

0.42

0.57

0.70

S.E.

0.16

0.13

0.18

0.23

N

9

10

10

9

EOS (%)

Mean

1.6

1.0*

1.1

1.5

% Difference

-37.5

-31.3

-6.3

S.D.

0.57

0.39

0.39

0.65

S.E.

0.19

0.12

0.12

0.22

N

9

10

10

9

BASO (%)

Mean

0.1

0.2

0.2

0.1

% Difference

100.0

100.0

0.0

S.D.

0.07

0.08

0.06

0.04

S.E.

0.02

0.03

0.02

0.01

N

9

10

10

9

LUC (%)

Mean

0.7

0.6

0.7

0.6

% Difference

-14.3

0.0

-14.3

S.D.

0.22

0.07

0.21

0.10

S.E.

0.07

0.02

0.06

0.03

N

9

10

10

9

NEU ABSOLUTE (thous/µL)

Mean

1.40

1.54

1.73

1.87

% Difference

10.0

23.6

33.6

S.D.

0.189

0.443

0.519

0.655

S.E.

0.063

0.140

0.164

0.218

N

9

10

10

9

LYMPH ABSOLUTE (thous/µL)

Mean

6.64

7.48

9.41**

6.90

% Difference

12.7

41.7

3.9

S.D.

1.180

0.975

1.998

1.310

S.E.

0.393

0.308

0.632

0.437

N

9

10

10

9

MONO ABSOLUTE (thous/µL)

Mean

0.18

0.19

0.28*

0.23

% Difference

5.6

55.6

27.8

S.D.

0.050

0.045

0.084

0.101

S.E.

0.017

0.014

0.027

0.034

N

9

10

10

9

EOS ABSOLUTE (thous/µL)

Mean

0.14

0.10

0.13

0.13

% Difference

-28.6

-7.1

-7.1

S.D.

0.047

0.035

0.046

0.053

S.E.

0.016

0.011

0.015

0.018

N

9

10

10

9

BASO ABSOLUTE (thous/µL)

Mean

0.01

0.01

0.02

0.01

% Difference

0.0

100.0

0.0

S.D.

0.008

0.008

0.009

0.004

S.E.

0.003

0.003

0.003

0.001

N

9

10

10

9

LUC ABSOLUTE (thous/µL)

Mean

0.06

0.05

0.08*

0.05

% Difference

-16.7

33.3

-16.7

S.D.

0.024

0.007

0.029

0.014

S.E.

0.008

0.002

0.009

0.005

N

9

10

10

9

RDW (%)

Mean

12.7

12.3

11.9**

11.1**

% Difference

-3.1

-6.3

-12.6

S.D.

0.42

0.28

0.31

0.57

S.E.

0.14

0.09

0.10

0.19

N

9

10

10

9

HDW (g/dL)

Mean

2.54

2.44

2.25**

1.95**

% Difference

-3.9

-11.4

-23.2

S.D.

0.143

0.184

0.113

0.080

S.E.

0.048

0.058

0.036

0.027

N

9

10

10

9

thous/µL = thousands/microliter

mil/µL = millions/microliter

fL = femtoliters

pg = picograms

g/dL = grams/deciliter

*   = significantly different from the control Group at 0.05 using Dunnett's test

** = significantly different from the control Group at 0.01 using Dunnett's test

Table S30 (Females)

Project no.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of Hematology And Coagulation Values

Females

Analysis Group:

0 mg/kg/Day

10 mg/kg/Day

25 mg/kg/Day

50 mg/kg/Day

WBC (thous/µL)

Mean

5.80

5.51

6.73

7.07

% Difference

-5.0

16.0

21.9

S.D.

2.676

2.026

2.077

1.536

S.E.

0.892

0.641

0.657

0.486

N

9

10

10

10

RBC (mil/µL)

Mean

8.19

8.66

8.50

7.86

% Difference

5.7

3.8

-4.0

S.D.

0.997

0.267

0.391

0.214

S.E.

0.332

0.084

0.124

0.068

N

9

10

10

10

HGB (g/dL)

Mean

15.2

15.5

15.5

14.6

% Difference

2.0

2.0

-3.9

S.D.

1.79

0.48

0.64

0.42

S.E.

0.60

0.15

0.20

0.13

N

9

10

10

10

HCT (%)

Mean

45.1

46.3

46.7

45.0

% Difference

2.7

3.5

-0.2

S.D.

4.81

1.37

1.86

1.63

S.E.

1.60

0.43

0.59

0.51

N

9

10

10

10

MCV (fL)

Mean

55.3

53.5

55.0

57.3*

% Difference

-3.3

-0.5

3.6

S.D.

1.96

1.54

1.49

1.53

S.E.

0.65

0.49

0.47

0.49

N

9

10

10

10

MCH (pg)

Mean

18.5

17.9*

18.2

18.6

% Difference

-3.2

-1.6

0.5

S.D.

0.49

0.53

0.49

0.61

S.E.

0.16

0.17

0.15

0.19

N

9

10

10

10

MCHC (g/dL)

Mean

33.5

33.4

33.2

32.4**

% Difference

-0.3

-0.9

-3.3

S.D.

0.77

0.28

0.57

0.63

S.E.

0.26

0.09

0.18

0.20

N

9

10

10

10

PLATELET (thous/µL)

Mean

951.

915.

933.

996.

% Difference

-3.8

-1.9

4.7

S.D.

179.3

125.3

178.1

128.8

S.E.

59.8

39.6

56.3

40.7

N

9

10

10

10

PT (seconds)

Mean

16.1

15.8

16.1

16.6

% Difference

-1.9

0.0

3.1

S.D.

0.69

0.65

0.47

0.55

S.E.

0.23

0.21

0.15

0.18

N

9

10

10

10

APTT (seconds)

Mean

13.6

13.6

12.7

12.2

% Difference

0.0

-6.6

-10.3

S.D.

1.15

1.52

2.13

1.84

S.E.

0.38

0.48

0.67

0.58

N

9

10

10

10

RETIC (%)

Mean

1.8

1.2

1.9

3.1*

% Difference

-33.3

5.6

72.2

S.D.

1.38

0.35

0.70

0.68

S.E.

0.46

0.11

0.22

0.22

N

9

10

10

10

RETIC ABSOLUTE (thous/µL)

Mean

137.7

105.1

161.4

240.3**

% Difference

-23.7

17.2

74.5

S.D.

65.65

31.41

56.56

57.77

S.E.

21.88

9.93

17.89

18.27

N

9

10

10

10

MPV (fL)

Mean

7.28

7.19

7.14

7.35

% Difference

-1.2

-1.9

1.0

S.D.

0.435

0.610

0.562

0.593

S.E.

0.145

0.193

0.178

0.188

N

9

10

10

10

NEU (%)

Mean

20.0

17.0

16.5

19.3

% Difference

-15.0

-17.5

-3.5

S.D.

7.53

7.91

4.63

2.99

S.E.

2.51

2.50

1.47

0.95

N

9

10

10

10

LYMPH (%)

Mean

76.0

78.6

79.3

76.5

% Difference

3.4

4.3

0.7

S.D.

7.72

8.03

5.00

3.08

S.E.

2.57

2.54

1.58

0.98

N

9

10

10

10

MONO (%)

Mean

2.2

2.2

1.9

2.5

% Difference

0.0

-13.6

13.6

S.D.

0.55

0.74

0.73

0.58

S.E.

0.18

0.23

0.23

0.18

N

9

10

10

10

EOS (%)

Mean

1.1

1.2

1.5

1.1

% Difference

9.1

36.4

0.0

S.D.

0.23

0.21

0.97

0.31

S.E.

0.08

0.07

0.31

0.10

N

9

10

10

10

BASO (%)

Mean

0.1

0.1

0.1

0.1

% Difference

0.0

0.0

0.0

S.D.

0.07

0.07

0.07

0.06

S.E.

0.02

0.02

0.02

0.02

N

9

10

10

10

LUC (%)

Mean

0.6

1.0*

0.6

0.6

% Difference

66.7

0.0

0.0

S.D.

0.21

0.37

0.23

0.14

S.E.

0.07

0.12

0.07

0.05

N

9

10

10

10

NEU ABSOLUTE (thous/µL)

Mean

1.21

0.86

1.07

1.38

% Difference

-28.9

-11.6

14.0

S.D.

1.082

0.261

0.355

0.473

S.E.

0.361

0.083

0.112

0.150

N

9

10

10

10

LYMPH ABSOLUTE (thous/µL)

Mean

 4.35

 4..40

 5.38

 5.39

% Difference

1.1

 23.7

23.9

S.D.

1.769

1.919

1.822

1.063

S.E.

0.590

0.607

0.576

0.336

N

9

10

10

10

MONO ABSOLUTE (thous/µL)

Mean

0.13

0.12

0.13

0.17

% Difference

 - 7.7

0.0

30.8

S.D.

0.073

0.061

0.073

0.066

S.E.

0.024

0.019

0.023

0.021

N

9

10

10

10

EOS ABSOLUTE (thous/µL)

Mean

0.06

0.07

0.09

0.08

% Difference

16.7

50.0

33.3

S.D.

0.034

0.020

0.040

0.020

S.E.

0.011

0.006

0.013

0.006

N

9

10

10

10

BASO ABSOLUTE (thous/µL)

Mean

0.00

0.01

0.01

0.01

% Difference

NA

NA

NA

S.D.

0.005

0.007

0.007

0.006

S.E.

0.002

0.002

0.002

0.002

N

9

10

10

10

LUC ABSOLUTE (thous/µL)

Mean

0.04

0.05

0.05

0.04

% Difference

25.0

25.0

0.0

S.D.

0.026

0.023

0.025

0.016

S.E.

0.009

0.007

0.008

0.005

N

9

10

10

10

RDW (%)

Mean

11.8

11.2

11.1

10.8**

% Difference

-5.1

-5.9

-8.5

S.D.

1.29

0.19

0.25

0.42

S.E.

0.43

0.06

0.08

0.13

N

9

10

10

10

HDW (g/dL)

Mean

2.17

2.10

2.00**

2.00**

% Difference

-3.2

-7.8

-7.8

S.D.

0.113

0.106

0.074

0.102

S.E.

0.038

0.033

0.023

0.032

N

9

10

10

10

thous/µL = thousands/microliter

mil/µL = millions/microliter

fL = femtoliters

pg = picograms

g/dL = grams/deciliter

*   = significantly different from the control Group at 0.05   using Dunnett's test

** = significantly different from the control Group at 0.01   using Dunnett's test

NA = not applicable

Table S31 (Males)

Project no.:WIL-387065M

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of Serum Chemistry Values

Males

Analysis Group:

0 mg/kg/Day

7 mg/kg/Day

15 mg/kg/Day

30 mg/kg/Day

ALBUMIN (g/dL)

Mean

4.1

4.1

4.4*

4.1

% Difference

0.0

7.3

0.0

S.D.

0.34

0.31

0.30

0.20

S.E.

0.11

0.10

0.09

0.06

N

10

10

10

10

Total Protein (g/dL)

Mean

6.7

6.7

7.2

6.8

% Difference

0.0

7.5

6.8

S.D.

0.62

0.45

0.48

0.33

S.E.

0.20

0.14

0.15

0.10

N

10

10

10

10

Globulin (g/dL)

Mean

2.7

2.6

2.8

2.6

% Difference

 -3.7

3.7

- 3.7

S.D.

0.33

0.16

0.23

0.17

S.E.

0.11

0.05

0.07

0.05

N

10

10

10

10

A/G Ratio

Mean

1.53

1.57

1.62

1.57

% Difference

2.6

5.9

2.6

S.D.

0.142

0.082

0.123

0.082

S.E.

0.045

0.026

0.039

0.026

N

10

10

10

10

Total BILI (mg/dL)

Mean

0.01

0.00

0.02

0.09**

% Difference

-100.0

100.0

800.0

S.D.

0.032

0.000

0.042

0.032

S.E.

0.010

0.000

0.013

0.010

N

10

10

10

10

Urea Nitrogen (mg/dL)

Mean

9.1

9.5

8.8

8.6

% Difference

4.4

-3.3

-5.5

S.D.

1.79

1.75

1.21

2.32

S.E.

0.57

0.55

0.38

0.73

N

10

10

10

10

Creatinine (mg/dL)

Mean

0.32

0.32

0.34

0.31

% Difference

0.0

6.3

-3.1

S.D.

0.057

0.079

0.058

0.046

S.E.

0.018

0.025

0.018

0.015

N

10

10

10

10

ALP (U/L)

Mean

231.

172.

190.

237.

% Difference

-25.5

-17.7

2.6

S.D.

89.0

64.7

68.9

93.0

S.E.

28.2

20.5

21.8

29.4

N

10

10

10

10

ALT (U/L)

Mean

36.

32.

39.

42.

% Difference

-11.1

8.3

16.7

S.D.

8.5

5.7

10.9

6.6

S.E.

2.7

1.8

3.4

2.1

N

10

10

10

10

AST (U/L)

Mean

94.

90.

101.

119.*

% Difference

-4.3

7.4

26.6

S.D.

19.5

16.5

15.6

29.4

S.E.

6.2

5.2

4.9

9.3

N

10

10

10

10

GGT (U/L)

Mean

0.0

0.0

0.0

0.0

% Difference

NA

NA

NA

S.D.

0.00

0.00

0.00

0.00

S.E.

0.00

0.00

0.00

0.00

N

10

10

10

10

Glucose (mg/dL)

Mean

113.

105.

109.

103.

% Difference

-7.1

-3.5

-8.8

S.D.

7.8

7.9

8.5

10.7

S.E.

2.5

2.5

2.7

3.4

N

10

10

10

10

Cholesterol (mg/dL)

Mean

62.

50.*

40.**

38.**

% Difference

-19.4

-35.5

-38.7

S.D.

15.6

6.5

7.8

4.6

S.E.

4.9

2.1

2.5

1.4

N

10

10

10

10

Calcium (mg/dL)

Mean

10.6

10.8

11.0

10.7

% Difference

1.9

3.8

0.9

S.D.

0.60

0.36

0.62

0.49

S.E.

0.19

0.11

0.20

0.15

N

10

10

10

10

Chloride (mEq/L)

Mean

105.

106.

106.

106.

% Difference

1.0

1.0

1.0

S.D.

1.3

1.1

1.0

1.9

S.E.

0.4

0.3

0.3

0.6

N

10

10

10

10

Phosphorus (mg/dL)

Mean

7.6

7.6

7.5

7.4

% Difference

0.0

-1.3

-2.6

S.D.

0.87

1.05

0.90

0.72

S.E.

0.28

0.33

0.29

0.23

N

10

10

10

10

Potassium (mEq/L)

Mean

5.92

5.66

5.74

5.47

% Difference

-4.4

-3.0

-7.6

S.D.

0.740

0.509

0.578

0.460

S.E.

0.234

0.161

0.183

0.145

N

10

10

10

10

Sodium (mEq/L)

Mean

147.

146.

147.

146.

% Difference

-0.7

0.0

-0.7

S.D.

1.8

2.9

2.2

1.5

S.E.

0.6

0.9

0.7

0.5

N

10

10

10

10

SDH (U/L)

Mean

9.

6.

9.

8.

% Difference

-33.3

0.0

-11.1

S.D.

5.0

2.7

4.5

3.2

S.E.

1.7

0.8

1.4

1.1

N

9

10

10

9

Triglyceride (mg/dL)

Mean

68.

74.

70.

78.

% Difference

8.8

2.9

14.7

S.D.

22.2

29.1

18.9

40.8

S.E.

7.0

9.2

6.0

12.9

N

10

10

10

10

mg/dL = milligrams/deciliter

U/L = international Unit/Liter

 g/dL = grams/deciliter

mEq/L = milliequivalents/Liter

*   = significantly different from the control Group at 0.05   using Dunnett's test

** = significantly different from the control Group at 0.01   using Dunnett's test

NA = not applicable

Table S32 (Females)

Project no.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of Serum Chemistry Values

Females

Analysis Group:

0 mg/kg/Day

10 mg/kg/Day

25 mg/kg/Day

50 mg/kg/Day

Albumin (g/dL)

Mean

4.6

4.6

4.6

4.4

% Difference

0.0

0.0

-4.3

S.D.

0.41

0.39

0.21

0.20

S.E.

0.14

0.12

0.07

0.06

N

9

10

10

10

Total Protein (g/dL)

Mean

7.3

7.3

7.4

7.2

% Difference

0.0

1.4

-1.4

S.D.

0.58

0.64

0.38

0.38

S.E.

0.19

0.20

0.12

0.12

N

9

10

10

10

Globulin (g/dL)

Mean

2.7

2.7

2.7

2.7

% Difference

0.0

0.0

0.0

S.D.

0.21

0.28

0.21

0.23

S.E.

0.07

0.09

0.07

0.07

N

9

10

10

10

A/G Ratio

Mean

1.68

1.70

1.72

1.63

% Difference

1.2

2.4

-3.0

S.D.

0.120

0.105

0.132

0.116

S.E.

0.040

0.033

0.042

0.037

N

9

10

10

10

Total BILI (mg/dL)

Mean

0.08

0.07

0.11

0.15*

% Difference

-12.5

37.5

87.5

S.D.

0.067

0.048

0.057

0.053

S.E.

0.022

0.015

0.018

0.017

N

9

10

10

10

Urea Nitrogen (mg/dL)

Mean

9.4

10.9

10.0

10.6

% Difference

16.0

6.4

12.8

S.D.

1.36

2.12

2.28

2.34

S.E.

0.45

0.67

0.72

0.74

N

9

10

10

10

Creatinine (mg/dL)

Mean

0.34

0.39

0.37

0.40

% Difference

14.7

8.8

17.6

S.D.

0.046

0.073

0.042

0.048

S.E.

0.015

0.023

0.013

0.015

N

9

10

10

10

ALP (U/L)

Mean

93.

75.

97.

133.

% Difference

-19.4

4.3

43.0

S.D.

40.5

24.7

30.4

46.7

S.E.

13.5

7.8

9.6

14.8

N

9

10

10

10

ALT (U/L)

Mean

43.

32.

28.

34.

% Difference

-25.6

-34.9

-20.9

S.D.

54.5

12.0

3.6

4.6

S.E.

18.2

3.8

1.1

1.5

N

9

10

10

10

AST (U/L)

Mean

103.

96.

88.

107.

% Difference

-6.8

-14.6

3.9

S.D.

50.3

12.3

12.8

12.6

S.E.

16.8

3.9

4.1

4.0

N

9

10

10

10

GGT (U/L)

Mean

0.3

0.1

0.2

0.3

% Difference

-66.7

-33.3

0.0

S.D.

0.50

0.32

0.42

0.48

S.E.

0.17

0.10

0.13

0.15

N

9

10

10

10

Glucose (mg/dL)

Mean

101.

108.

103.

97.

% Difference

6.9

2.0

-4.0

S.D.

7.9

9.8

10.3

9.7

S.E.

2.6

3.1

3.3

3.1

N

9

10

10

10

Cholesterol (mg/dL)

Mean

66.

66.

45.**

43.**

% Difference

0.0

-31.8

-34.8

S.D.

9.3

12.3

7.3

10.6

S.E.

3.1

3.9

2.3

3.3

N

9

10

10

10

Calcium (mg/dL)

Mean

11.1

10.9

10.9

10.9

% Difference

-1.8

-1.8

-1.8

S.D.

0.47

0.66

0.27

0.27

S.E.

0.16

0.21

0.08

0.08

N

9

10

10

10

Chloride (mEq/L)

Mean

105.

107.

106.

106.

% Difference

1.9

1.0

1.0

S.D.

1.1

1.4

1.5

2.0

S.E.

0.4

0.4

0.5

0.6

N

9

10

10

10

Phosphorus (mg/dL)

Mean

6.2

6.0

6.5

6.9

% Difference

-3.2

4.8

11.3

S.D.

0.59

0.56

0.81

0.81

S.E.

0.20

0.18

0.26

0.26

N

9

10

10

10

Potassium (mEq/L)

Mean

5.19

5.08

5.10

4.99

% Difference

-2.1

-1.7

-3.9

S.D.

0.559

0.181

0.323

0.432

S.E.

0.186

0.057

0.102

0.137

N

9

10

10

10

Sodium (mEq/L)

Mean

145.

145.

144.

144.

% Difference

0.0

-0.7

-0.7

S.D.

1.1

0.7

0.8

1.1

S.E.

0.4

0.2

0.3

0.4

N

9

10

10

10

SDH (U/L)

Mean

16.

8.

8.

10.

% Difference

-50.0

-50.0

-37.5

S.D.

27.9

6.5

4.0

4.8

S.E.

9.3

2.1

1.3

1.5

N

9

10

10

10

Triglyceride (mg/dL)

Mean

32.

38.

29.

42.

% Difference

18.8

-9.4

31.3

S.D.

10.3

15.7

7.3

23.6

S.E.

3.4

5.0

2.3

7.5

N

9

10

10

10

mg/dL = milligrams/deciliter

U/L = international Unit/Liter

 g/dL = grams/deciliter

mEq/L = milliequivalents/Liter

* = significantly different from the control Group at 0.05 using Dunnett's test

** = significantly different from the control Group at 0.01 using Dunnett's test

NA = not applicable

Table S39 (Unscheduled Death - Females)

Project no.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of Macroscopic Findings

Found Dead or Euthanized Moribund or in Extremis

Female

 Group:

1

2

3

4

Number of Animals Examined

1

0

0

0

Lungs

 -not fully collapsed

1

0

0

0

Skin

 -matting, red

1

0

0

0

Thymus

 -Area(s), Dark red

1

0

0

0

Uterus

 -Contents,  Clear Fluid

1

0

0

0

1 - 0 mg/kg/Day

2 - 10 mg/kg/Day

3 - 25 mg/kg/Day

4 - 50 mg/kg/Day

Table S42 (Scheduled Necropsy - Males)

Project no.:WIL-387065M

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of ORGAN WEIGHTS And RELATIVE ORGAN WEIGHTS

Males

Group:

0 mg/kg/Day

7 mg/kg/Day

15 mg/kg/Day

30 mg/kg/Day

Final Body WT (g)

Mean

559.

551.

567.

505.

% Difference

-1.4

1.4

-9.7

S.D.

74.2

51.6

43.3

70.7

S.E.

23.5

16.3

13.7

22.4

N

10

10

10

10

Adrenal Glands (g)

Mean

0.0578

0.0562

0.0551

0.0607

% Difference

-2.8

-4.7

5.0

S.D.

0.00750

0.01041

0.00815

0.00758

S.E.

0.00237

0.00329

0.00258

0.00240

N

10

10

10

10

Adrenal Glands (g/100 g Final Body Weight)

Mean

0.010

0.010

0.010

0.012*

% Difference

0.0

0.0

20.0

S.D.

0.0008

0.0016

0.0013

0.0018

S.E.

0.0003

0.0005

0.0004

0.0006

N

10

10

10

10

Adrenal Glands (g/100 g Brain)

Mean

2.687

2.582

2.584

2.837

% Difference

-3.9

-3.8

5.6

S.D.

0.3348

0.4927

0.3787

0.3581

S.E.

0.1059

0.1558

0.1198

0.1132

N

10

10

10

10

Brain (g)

Mean

2.15

2.18

2.13

2.14

% Difference

1.4

-0.9

-0.5

S.D.

0.099

0.105

0.076

0.110

S.E.

0.031

0.033

0.024

0.035

N

10

10

10

10

Brain (g/100 g Final Body Weight)

Mean

0.389

0.398

0.378

0.430

% Difference

2.3

-2.8

10.5

S.D.

0.0378

0.0402

0.0276

0.0571

S.E.

0.0120

0.0127

0.0087

0.0181

N

10

10

10

10

Epididymides (g)

Mean

1.33

1.35

1.29

1.27

% Difference

1.5

-3.0

-4.5

S.D.

0.128

0.134

0.098

0.189

S.E.

0.040

0.042

0.031

0.060

N

10

10

10

10

Epididymides (g/100 g Final Body Weight)

Mean

0.240

0.246

0.229

0.253

% Difference

2.5

-4.6

5.4

S.D.

0.0294

0.0268

0.0292

0.0379

S.E.

0.0093

0.0085

0.0092

0.0120

N

10

10

10

10

Epididymides (g/100 g Brain)

Mean

61.636

62.043

60.516

59.257

% Difference

0.7

-1.8

-3.9

S.D.

4.5133

5.7205

4.2378

8.6504

S.E.

1.4272

1.8090

1.3401

2.7355

N

10

10

10

10

Heart (g)

Mean

1.63

1.77

1.75

1.73

% Difference

8.6

7.4

6.1

S.D.

0.194

0.239

0.117

0.221

S.E.

0.061

0.076

0.037

0.070

N

10

10

10

10

Heart (g/100 g Final Body Weight)

Mean

0.293

0.321

0.309

0.345**

% Difference

9.6

5.5

17.7

S.D.

0.0306

0.0334

0.0314

0.0340

S.E.

0.0097

0.0106

0.0099

0.0108

N

10

10

10

10

Heart (g/100 g Brain)

Mean

75.601

80.959

81.970

81.081

% Difference

7.1

8.4

7.2

S.D.

6.5112

10.0613

6.0525

9.7848

S.E.

2.0590

3.1817

1.9140

3.0942

N

10

10

10

10

Kidneys (g)

Mean

3.40

3.40

3.51

3.27

% Difference

0.0

3.2

-3.8

S.D.

0.398

0.351

0.362

0.413

S.E.

0.126

0.111

0.114

0.131

N

10

10

10

10

Kidneys (g/100 g Final Body Weight)

Mean

0.613

0.618

0.619

0.649

% Difference

0.8

1.0

5.9

S.D.

0.0702

0.0445

0.0526

0.0447

S.E.

0.0222

0.0141

0.0166

0.0141

N

10

10

10

10

Kidneys (g/100 g Brain)

Mean

157.784

156.144

164.482

152.695

% Difference

-1.0

4.2

-3.2

S.D.

14.2818

15.9263

14.1581

17.6914

S.E.

4.5163

5.0363

4.4772

5.5945

N

10

10

10

10

Liver (g)

Mean

16.22

17.02

19.12

18.22

% Difference

4.9

17.9

12.3

S.D.

2.057

2.078

2.314

3.820

S.E.

0.650

0.657

0.732

1.208

N

10

10

10

10

Liver (g/100 g Final Body Weight)

Mean

2.905

3.085

3.364**

3.583**

% Difference

6.2

15.8

23.3

S.D.

0.1449

0.1933

0.2313

0.2784

S.E.

0.0458

0.0611

0.0731

0.0880

N

10

10

10

10

Liver (g/100 g Brain)

Mean

751.589

780.740

895.968*

849.558

% Difference

3.9

19.2

13.0

S.D.

71.2294

86.0663

96.4590

154.1345

S.E.

22.5247

27.2166

30.5030

48.7416

N

10

10

10

10

*   = significantly different from the control Group at 0.05 using Dunnett's test

** = significantly different from the control Group at 0.01 using Dunnett's test

Table S42 (Scheduled Necropsy - Males) - continued

Project no.:WIL-387065M

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of ORGAN WEIGHTS And RELATIVE ORGAN WEIGHTS

Males

Group:

0 mg/kg/Day

7 mg/kg/Day

15 mg/kg/Day

30 mg/kg/Day

Putuitary (g)

Mean

0.0148

0.0150

0.0148

0.0142

% Difference

1.4

0.0

-4.1

S.D.

0.00176

0.00221

0.00149

0.00244

S.E.

0.00056

0.00070

0.00047

0.00077

N

10

10

10

10

Putuitary (g/100 g Final Body Weight)

Mean

0.003

0.003

0.003

0.003

% Difference

0.0

0.0

0.0

S.D.

0.0004

0.0004

0.0003

0.0004

S.E.

0.0001

0.0001

0.0001

0.0001

N

10

10

10

10

Putuitary (g/100 g Brain)

Mean

0.689

0.687

0.696

0.664

% Difference

-0.3

1.0

-3.6

S.D.

0.0837

0.0848

0.0648

0.1095

S.E.

0.0265

0.0268

0.0205

0.0346

N

10

10

10

10

Sem Ves/Prostate (g)

Mean

3.49

3.19

3.07*

3.16

% Difference

-8.6

-12.0

-9.5

S.D.

0.309

0.222

0.362

0.422

S.E.

0.098

0.070

0.114

0.133

N

10

10

10

10

Sem Ves/Prostate (g/100 g Final Body Weight)

Mean

0.633

0.581

0.544

0.634

% Difference

-8.2

-14.1

0.2

S.D.

0.0940

0.0497

0.0750

0.1087

S.E.

0.0297

0.0157

0.0237

0.0344

N

10

10

10

10

Sem Ves/Prostate (g/100 g Brain)

Mean

162.327

146.400

143.927

148.119

% Difference

-9.8

-11.3

-8.8

S.D.

15.8062

10.4675

15.2144

21.5858

S.E.

4.9983

3.3101

4.8112

6.8260

N

10

10

10

10

Spleen (g)

Mean

0.77

0.80

0.84

0.90

% Difference

3.9

9.1

16.9

S.D.

0.149

0.159

0.117

0.146

S.E.

0.047

0.050

0.037

0.046

N

10

10

10

10

Spleen (g/100 g Final Body Weight)

Mean

0.137

0.144

0.149

0.180**

% Difference

5.1

8.8

31.4

S.D.

0.0229

0.0229

0.0169

0.0264

S.E.

0.0073

0.0072

0.0053

0.0083

N

10

10

10

10

Spleen (g/100 g Brain)

Mean

35.467

36.680

39.536

42.074

% Difference

3.4

11.5

18.6

S.D.

6.0478

7.8153

5.2422

6.1934

S.E.

1.9125

2.4714

1.6577

1.9585

N

10

10

10

10

Testes (g)

Mean

3.65

3.58

3.51

3.19**

% Difference

-1.9

-3.8

-12.6

S.D.

0.320

0.277

0.252

0.443

S.E.

0.101

0.088

0.080

0.140

N

10

10

10

10

Testes (g/100 g Final Body Weight)

Mean

0.658

0.653

0.622

0.636

% Difference

-0.8

-5.5

-3.3

S.D.

0.0554

0.0666

0.0579

0.0945

S.E.

0.0175

0.0211

0.0183

0.0299

N

10

10

10

10

Testes (g/100 g Brain)

Mean

169.468

164.487

164.811

148.557**

% Difference

-2.9

-2.7

-12.3

S.D.

10.0073

15.5624

10.7851

16.4258

S.E.

3.1646

4.9213

3.4106

5.1943

N

10

10

10

10

Thymus (g)

Mean

0.2411

0.2383

0.2168

0.2214

% Difference

-1.2

-10.1

-8.2

S.D.

0.06370

0.08929

0.03920

0.03895

S.E.

0.02014

0.02824

0.01240

0.01232

N

10

10

10

10

Thymus (g/100 g Final Body Weight)

Mean

0.043

0.043

0.038

0.045

% Difference

0.0

-11.6

4.7

S.D.

0.0101

0.0152

0.0063

0.0106

S.E.

0.0032

0.0048

0.0020

0.0033

N

10

10

10

10

Thymus (g/100 g Brain)

Mean

11.219

10.905

10.160

10.395

% Difference

-2.8

-9.4

-7.3

S.D.

2.9692

3.9984

1.7240

2.0852

S.E.

0.9389

1.2644

0.5452

0.6594

N

10

10

10

10

Thyroids/Parathy (g)

Mean

0.0166

0.0151

0.0168

0.0156

% Difference

-9.0

1.2

-6.0

S.D.

0.00351

0.00224

0.00162

0.00142

S.E.

0.00111

0.00071

0.00051

0.00045

N

10

10

10

10

Thyroids/Parathy (g/100 g Final Body Weight)

Mean

0.003

0.003

0.003

0.003

% Difference

0.0

0.0

0.0

S.D.

0.0005

0.0005

0.0000

0.0007

S.E.

0.0001

0.0002

0.0000

0.0002

N

10

10

10

10

Thyroids/Parathy (g/100 g Brain)

Mean

0.769

0.696

0.785

0.730

% Difference

-9.5

2.1

-5.1

S.D.

0.1528

0.1163

0.0634

0.0817

S.E.

0.0483

0.0368

0.0200

0.0258

N

10

10

10

10

*   = significantly different from the control Group at 0.05 using Dunnett's test

** = significantly different from the control Group at 0.01 using Dunnett's test

Table S43  (Scheduled Necropsy - Females)

Project no.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of Organ Weights And Relative Organ Weights

Females

Group:

0 mg/kg/Day

10 mg/kg/Day

25 mg/kg/Day

50 mg/kg/Day

Final Body WT (g)

Mean

287.

289.

293.

306.

% Difference

0.7

2.1

6.6

S.D.

24.7

21.6

21.8

32.0

S.E.

8.2

6.8

6.9

10.1

N

9

10

10

10

Adrenal Glands (g)

Mean

0.0678

0.0739

0.0734

0.0843

% Difference

9.0

8.3

24.3

S.D.

0.00960

0.01121

0.01876

0.01017

S.E.

0.00320

0.00354

0.00593

0.00322

N

9

10

10

10

Adrenal Glands (g/100 g Final Body Weight)

Mean

0.024

0.026

0.025

0.028

% Difference

8.3

4.2

16.7

S.D.

0.0038

0.0032

0.0069

0.0029

S.E.

0.0013

0.0010

0.0022

0.0009

N

9

10

10

10

Adrenal Glands (g/100 g Brain)

Mean

3.406

3.709

3.718

4.140

% Difference

8.9

9.2

21.6

S.D.

0.5106

0.5761

0.9297

0.5144

S.E.

0.1702

0.1822

0.2940

0.1627

N

9

10

10

10

Brain (g)

Mean

1.99

1.99

1.97

2.04

% Difference

0.0

-1.0

2.5

S.D.

0.046

0.049

0.056

0.058

S.E.

0.015

0.016

0.018

0.018

N

9

10

10

10

Brain (g/100 g Final Body Weight)

Mean

0.699

0.694

0.677

0.670

% Difference

-0.7

-3.1

-4.1

S.D.

0.0685

0.0571

0.0427

0.0581

S.E.

0.0228

0.0181

0.0135

0.0184

N

9

10

10

10

Heart (g)

Mean

1.06

1.08

1.10

1.23**

% Difference

1.9

3.8

16.0

S.D.

0.072

0.083

0.070

0.164

S.E.

0.024

0.026

0.022

0.052

N

9

10

10

10

Heart (g/100 g Final Body Weight)

Mean

0.372

0.373

0.376

0.400

% Difference

0.3

1.1

7.5

S.D.

0.0313

0.0195

0.0288

0.0258

S.E.

0.0104

0.0062

0.0091

0.0082

N

9

10

10

10

Heart (g/100 g Brain)

Mean

53.409

53.967

55.585

60.142*

% Difference

1.0

4.1

12.6

S.D.

3.9906

3.9913

4.3138

7.2659

S.E.

1.3302

1.2621

1.3641

2.2977

N

9

10

10

10

Kidneys (g)

Mean

1.93

1.90

1.96

2.06

% Difference

-1.6

1.6

6.7

S.D.

0.168

0.132

0.119

0.242

S.E.

0.056

0.042

0.038

0.077

N

9

10

10

10

Kidneys (g/100 g Final Body Weight)

Mean

0.674

0.660

0.671

0.671

% Difference

-2.1

-0.4

-0.4

S.D.

0.0468

0.0266

0.0391

0.0392

S.E.

0.0156

0.0084

0.0124

0.0124

N

9

10

10

10

Kidneys (g/100 g Brain)

Mean

97.101

95.506

99.256

100.769

% Difference

-1.6

2.2

3.8

S.D.

10.0797

6.4723

4.5140

10.3560

S.E.

3.3599

2.0467

1.4275

3.2748

N

9

10

10

10

Liver (g)

Mean

8.88

9.31

9.86

11.60**

% Difference

4.8

11.0

30.6

S.D.

0.991

0.908

1.075

1.558

S.E.

0.330

0.287

0.340

0.493

N

9

10

10

10

Liver (g/100 g Final Body Weight)

Mean

3.093

3.220

3.363**

3.778**

% Difference

4.1

8.7

22.1

S.D.

0.1445

0.1459

0.1803

0.1466

S.E.

0.0482

0.0461

0.0570

0.0464

N

9

10

10

10

Liver (g/100 g Brain)

Mean

446.686

467.806

498.900

568.529**

% Difference

4.7

11.7

27.3

S.D.

55.8171

51.5812

45.1091

66.4082

S.E.

18.6057

16.3114

14.2647

21.0001

N

9

10

10

10

* = significantly different from the control Group at 0.05 using Dunnett's test

** = significantly different from the control Group at 0.01 using Dunnett's test

Table S43  (Scheduled Necropsy - Females) - continued

Project no.:WIL-387065F

A 90-Day Oral Study of N-Butyronitrile in Rats

Sponsor: EASTMAN CHEMICAL CO.

Summary of Organ Weights And Relative Organ Weights

Females

Group:

0 mg/kg/Day

10 mg/kg/Day

25 mg/kg/Day

50 mg/kg/Day

Ovaries/Oviducts (g)

Mean

0.1199

0.1202

0.1339

0.1604**

% Difference

0.3

11.7

33.8

S.D.

0.01769

0.03089

0.01729

0.02599

S.E.

0.00590

0.00977

0.00547

0.00822

N

9

10

10

10

Ovaries/Oviducts (g/100 g Final Body Weight)

Mean

0.042

0.042

0.046

0.052*

% Difference

0.0

9.5

23.8

S.D.

0.0075

0.0095

0.0058

0.0061

S.E.

0.0025

0.0030

0.0018

0.0019

N

9

10

10

10

Ovaries/Oviducts (g/100 g Brain)

Mean

6.020

6.019

6.782

7.868**

% Difference

0.0

12.7

30.7

S.D.

0.8847

1.5179

0.8228

1.2355

S.E.

0.2949

0.4800

0.2602

0.3907

N

9

10

10

10

Putuitary (g)

Mean

0.0197

0.0181

0.0188

0.0174

% Difference

-8.1

-4.6

-11.7

S.D.

0.00227

0.00336

0.00272

0.00377

S.E.

0.00076

0.00106

0.00086

0.00119

N

9

10

10

10

Putuitary (g/100 g Final Body Weight)

Mean

0.007

0.006

0.006

0.006

% Difference

-14.3

-14.3

-14.3

S.D.

0.0008

0.0010

0.0010

0.0010

S.E.

0.0003

0.0003

0.0003

0.0003

N

9

10

10

10

Putuitary g/100 g Brain)

Mean

0.988

0.907

0.954

0.854

% Difference

-8.2

-3.4

-13.6

S.D.

0.1153

0.1738

0.1326

0.1801

S.E.

0.0384

0.0549

0.0419

0.0570

N

9

10

10

10

Spleen (g)

Mean

0.51

0.53

0.61

0.81**

% Difference

3.9

19.6

58.8

S.D.

0.077

0.059

0.119

0.136

S.E.

0.026

0.019

0.038

0.043

N

9

10

10

10

Spleen (g/100 g Final Body Weight)

Mean

0.180

0.182

0.208

0.263**

% Difference

1.1

15.6

46.1

S.D.

0.0307

0.0123

0.0322

0.0334

S.E.

0.0102

0.0039

0.0102

0.0106

N

9

10

10

10

Spleen (g/100 g Brain)

Mean

25.838

26.446

30.805

39.466**

% Difference

2.4

19.2

52.7

S.D.

3.8775

3.0220

5.3292

6.0115

S.E.

1.2925

0.9556

1.6852

1.9010

N

9

10

10

10

Thymus (g)

Mean

0.2163

0.2125

0.2409

0.2522

% Difference

-1.8

11.4

16.6

S.D.

0.04576

0.04504

0.02670

0.06149

S.E.

0.01525

0.01424

0.00844

0.01944

N

9

10

10

10

Thymus (g/100 g Final Body Weight)

Mean

0.076

0.074

0.083

0.083

% Difference

-2.6

9.2

9.2

S.D.

0.0195

0.0149

0.0143

0.0245

S.E.

0.0065

0.0047

0.0045

0.0077

N

9

10

10

10

Thymus (g/100 g Brain)

Mean

10.849

10.640

12.235

12.375

% Difference

-1.9

12.8

14.1

S.D.

2.2090

2.1434

1.5932

3.0441

S.E.

0.7363

0.6778

0.5038

0.9626

N

9

10

10

10

Thyroids/Parathy (g)

Mean

0.0144

0.0137

0.0137

0.0147

% Difference

-4.9

-4.9

2.1

S.D.

0.00280

0.00314

0.00221

0.00226

S.E.

0.00093

0.00099

0.00070

0.00071

N

9

10

10

10

Thyroids/Parathy (g/100 g Final Body Weight)

Mean

0.005

0.005

0.005

0.005

% Difference

0.0

0.0

0.0

S.D.

0.0011

0.0013

0.0008

0.0008

S.E.

0.0004

0.0004

0.0003

0.0003

N

9

10

10

10

Thyroids/Parathy (g/100 g Brain)

Mean

0.719

0.687

0.696

0.721

% Difference

-4.5

-3.2

0.3

S.D.

0.1322

0.1554

0.1106

0.1116

S.E.

0.0441

0.0492

0.0350

0.0353

N

9

10

10

10

Uterus (g)

Mean

0.71

0.73

0.73

0.68

% Difference

2.8

2.8

-4.2

S.D.

0.178

0.271

0.172

0.256

S.E.

0.059

0.086

0.054

0.081

N

9

10

10

10

Uterus (g/100 g Final Body Weight)

Mean

0.248

0.253

0.249

0.223

% Difference

2.0

0.4

-10.1

S.D.

0.0582

0.0988

0.0596

0.0842

S.E.

0.0194

0.0312

0.0189

0.0266

N

9

10

10

10

Uterus (g/100 g Brain)

Mean

35.707

36.226

36.905

33.384

% Difference

1.5

3.4

-6.5

S.D.

9.3551

12.6223

9.1697

13.0704

S.E.

3.1184

3.9915

2.8997

4.1332

N

9

10

10

10

* = significantly different from the control Group at 0.05 using Dunnett's test

** = significantly different from the control Group at 0.01 using Dunnett's test

Discussion:

The objective of the study was to evaluate the potential toxicity of n-Butyronitrile when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days. This study also included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

Test substance-related findings included clinical observations of wet clear material on various body surfaces, including the mouth, neck, and forelimbs as well as salivation. These observations were present in the 15 and 30 mg/kg/day group males and the 25 and/or 50 mg/kg/day group females in a dose responsive manner, and at a very limited incidence in the 7 mg/kg/day group males and 10 mg/kg/day group females.

Lower body weights in the 30 mg/kg/day group males due to lower body weight gains were observed at study week 3 and continued through the remainder of the study. Although body weights in the 30 mg/kg/day group males were minimally lower than the concurrent control group (≤7.5%), the cumulative body weight gain of the 30 mg/kg/day group at the end of the dosing period (study week 0 to 13) was 12.4% less than that of the concurrent control group.

Relationships were suspected between test substance-related organ weight, clinical pathology, and/or histopathology observations. These proposed relationships were based on subjective interpretation rather than a statistical analysis of correlation.

Increased liver weights, which may be an adaptive response rather than an adverse response, were observed in the 25 and 50 mg/kg/day group females. As hepatocellular hypertrophy was not observed, this change was correlated with the finding of hepatocellular cytoplasmic rarefaction in the 50 mg/kg/day group. However, hepatocellular hypertrophy is difficult to assess microscopically if the increase in hepatocyte size uniformly affects the entire liver lobule.

Therefore, a lack of a microscopic diagnosis of hepatocellular hypertrophy does not rule out the existence of enlarged hepatocytes.

The hematologic changes including higher reticulocyte count and MCV, and lower MCHC in the 30 mg/kg/day group males and 50 mg/kg/day group females are consistent with a regenerative hematologic response. There were no gross or histologic findings suggestive of internal or external hemorrhage; however, several observations were suggestive of increased red blood cell turnover or hemolysis. These changes included an increase in AST and serum bilirubin, suggestive of erythrocyte damage and increased hemoglobin turnover, as well the presence of brown pigment (hemosiderin, presumptive) within macrophages in the splenic red pulp in the 30 mg/kg/day group males, suggestive of hemosiderin deposition following red blood cell death (hemolysis).

Although the presence of brown pigment was also noted in the spleen of the 7 and 15 mg/kg/day group males, this change was not associated with any hematologic or serum chemistry changes and, as such, was not considered adverse at 7 or 15 mg/kg/day.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, oral administration of n-Butyronitrile to Sprague Dawley rats at dosage levels of 7, 15, and 30 mg/kg/day in males and 10, 25, and 50 mg/kg/day in females for a minimum of 90 days was tolerated at all doses. Test substance-related reductions in male body weights, hematology parameters (higher reticulocytes and MCV; lower MCHC), serum chemistry parameters (higher total bilirubin and AST), higher heart, liver, and spleen weights and/or histologic findings (accumulation of pigment in the spleen and hepatocellular cytoplasmic rarefaction in the liver) were observed at 30 mg/kg/day in males and 50 mg/kg/day in females. As a result, the No-Observed-Adverse-Effect-Level (NOAEL) is 15 mg/kg/day in the males and 25 mg/kg/day in the females.
Executive summary:

The objective of the study was to evaluate the potential toxicity of n-Butyronitrile when administered daily by oral gavage to Sprague Dawley rats for a minimum of 90 consecutive days. This study also included evaluation of potential neurotoxicity by functional observational battery (FOB) and motor activity (MA) assessment.

 

n-Butyronitrile in the vehicle (corn oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to 3 toxicology groups (Groups 2-4) of Crl:CD(SD) rats. Dosage levels were 7, 15, and 30 mg/kg/day for Groups 2, 3, and 4 for the males, respectively, and the dosage levels were 10, 25, and 50 mg/kg/day for Groups 2, 3, and 4 for the females, respectively. A concurrent control group (Group 1) received the vehicle (corn oil) on a comparable regimen. The dose volume was 10 mL/kg for all groups. All groups consisted of 10 animals/sex. Following 90 or 91 days of dose administration, all animals were euthanized For toxicology assessment, all animals were observed twice daily for mortality and moribundity. Clinical examinations were performed daily, and detailed physical examinations were performed weekly (± 2 days). Individual body weights were recorded weekly (± 2 days). Cage food weights were recorded once weekly (± 2 days) beginning following randomization. Functional observational battery (FOB) and motor activity data were recorded for all animals during the final week of the dosing period (study week 12). Ophthalmic examinations were performed during study week -2 for all animals, and study week 12 for all surviving animals in Groups 1 and 4 only. Clinical pathology parameters (hematology, coagulation, serum chemistry, and urinalysis) were analyzed for all surviving animals on the day of the scheduled necropsy. Complete necropsies were conducted on all animals, and selected organs were weighed at the scheduled necropsy. Selected tissues were examined microscopically from the animal euthanizedin extremisand all animals in the control and high-dose groups at the scheduled necropsy. In addition, the liver was identified as a potential target tissue and was examined microscopically from all animals, while the spleen was identified as a potential target tissue in males only and was examined from all male animals. Gross lesions were examined microscopically from all animals.

 

There were no test substance-related effects on survival, food consumption, coagulation or urinalysis parameters. There were no test substance-related ophthalmic or macroscopic findings. FOB and MA assessments were unaffected by test substance administration. Test substance-related clinical observations included wet clear material on various body surfaces, including the mouth, neck, trunk, and forelimbs as well as salivation. These observations were present in the high- and/or mid-dose group males and females in a dose-related manner. These observations were also noted at a very limited incidence in the low-dose group males and females. Test substance-related lower body weight gains in the 30 mg/kg/day group males throughout the study resulted in lower body weights observed in the 30 mg/kg/day group males from study week 3 through the remainder of the study. Test substance-related clinical pathology changes included higher absolute reticulocyte values and lower mean corpuscular hemoglobin concentration (MCHC) in the 30 mg/kg/day group males and 50 mg/kg/day group females; higher mean corpuscular volume (MCV) values in the 50 mg/kg/day group females; higher total bilirubin (TBIL) values in the 30 mg/kg/day group males and 50 mg/kg/day group females; and higher aspartate aminotransferase (AST) values in the 30 mg/kg/day group males. Lower cholesterol values were also noted in the 7, 15, and 30 mg/kg/day group males and 25 and 50 mg/kg/day group females which was considered test-substance related, but likely toxicologically irrelevant. Test substance-related higher heart, liver, and spleen weights were noted in the 25 and/or 50 mg/kg/day group females. Test substance-related histologic findings included accumulation of pigment in the spleen of the 7, 15, and 30 mg/kg/day group males and hepatocellular cytoplasmic rarefaction in the liver of the 7, 15, and 30 mg/kg/day group males and 25 and 50 mg/kg/day group females.

 

Based on the results of this study, oral administration of n-Butyronitrile to Sprague Dawley rats at dosage levels of 7, 15, and 30 mg/kg/day in males and 10, 25, and 50 mg/kg/day in females for a minimum of 90 days was tolerated at all doses. Test substance-related reductions in male body weights, hematology parameters (higher reticulocytes and MCV; lower MCHC), serum chemistry parameters (higher total bilirubin and AST), higher heart, liver, and spleen weights and/or histologic findings (accumulation of pigment in the spleen and hepatocellular cytoplasmic rarefaction in the liver) were observed at 30 mg/kg/day in males and 50 mg/kg/day in females. As a result, the No-Observed-Adverse-Effect-Level (NOAEL) is 15 mg/kg/day in the males and 25 mg/kg/day in the females.