4-phenylbutenone

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This information comes from an NTP-report (TR 572). The quality of this report is considered to be high.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Principles of method if other than guideline:

Male and female F344/N rats and B6C3F1 mice received methyl trans-styryl ketone (98.6 % pure) in feed for 3 month and dermally for 3 month or 2 years. Two-year studies were conducted to provide data for assessment of possible toxicity due to exposure to methyl trans-styryl ketone. The dermal route was chosen since this is the route for highest human exposure and due to studies demonstrating systemic exposure following dermal application to methyl trans-styryl ketone.

GLP compliance:

not specified

Remarks:

It is not stated in the publication but it can be assumed, that the test was conducted according to GLP criteria.

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

methyl trans-styryl ketone

IUPAC Name:

methyl trans-styryl ketone

Constituent 2

Reference substance name:

trans-4-phenylbut-3-en-2-one

EC Number:

217-587-6

EC Name:

trans-4-phenylbut-3-en-2-one

Cas Number:

1896-62-4

Molecular formula:

C10H10O

IUPAC Name:

4-phenylbut-3-en-2-one

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): methyl trans-styryl ketone
- Molecular formula (if other than submission substance): C10H10O
- Molecular weight (if other than submission substance): 146.19
- Structural formula attached as image file (if other than submission substance): see Fig. 1
- Substance type: organic
- Physical state: a solid, with a white to yellow in color and with a sweet, pungent, creamy, floral odor
- Analytical purity: 98.6 % pure
- Purity test: Identity, purity, and stability analyses were conducted by the analytical chemistry laboratory at Battelle Columbus Operations (Columbus, OH). Identity and purity analyses were conducted by the study laboratories at BioReliance Corporation (Rockville, MD; 3-month studies)
- Lot/batch No.: Lot 21805LN
- Stability under test conditions: Stability studies of the bulk chemical were performed by the analytical chemistry laboratory using GC. These studies indicated that methyl trans-styryl ketone was stable as a bulk chemical for at least 14 days when stored in sealed amber glass containers at temperatures up to 25° C. To ensure stability, the bulk chemical was stored at room temperature, protected from light and moisture under a nitrogen headspace in amber glass bottles sealed with Teflon®-lined lids. Periodic reanaly-ses of the bulk chemical were performed by the study laboratory approximately every 6 months during the 2-year studies using GC; no degradation of the bulk chemical was detected.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY)
- Age at study initiation: On receipt, the rats and mice were approximately 4 weeks old. Rats were quarantined for 11 (males) or 12 (females) days and were 5 to 6 weeks old on the first day of the study.
- Housing: Rats and female mice were housed five per cage and male mice were housed individually.
- Diet (e.g. ad libitum): Feed was available ad libitum (Irradiated NTP-2000 meal diet)
- Water (e.g. ad libitum): Water was available ad libitum (Tap water via automatic watering system)
- Acclimation period: 11 / 12 days of quaratine

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72° ± 3° F
- Humidity (%): 50% ± 15%
- Air changes (per hr): at least 10/hour
- Photoperiod (hrs dark / hrs light): 12 hours/day

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
PREPARATION AND ANALYSIS OF DOSE FORMULATIONS
The dose formulations were prepared five times by mixing methyl trans-styryl ketone with feed. A premix was prepared by hand and then blended with additional feed in a Patterson-Kelly twin-shell blender for 15 minutes using an intensifier bar for the initial 5 minutes. The dose formulations were stored in double polyethylene bags with twist-ties at room temperature for up to 48 days.
Homogeneity studies of 0.03125% and 0.5% formula-tions and stability studies of 0.005% and 0.03125% formulations were performed by the analytical chemistry laboratory using GC. Additional homogeneity studies of the 0.025% and 0.4% dose formulations were performed by the study laboratory using GC. Homogeneity was confirmed, and stability was confirmed for at least 48 days for dose formulations stored in sealed plastic bags protected from light at room temperature and below, and for at least 7 days under simulated animal room conditions if the dosed feed was kept free from contamination with rodent urine and feces.
Periodic analyses of the dose formulations of methyl trans-styryl ketone were conducted by the study laboratory using GC. The dose formulations were analyzed three times; animal room samples of these dose formu-lations were also analyzed. Of the dose formulations analyzed, 15 of 17 for rats and mice were within 10% of the target concentrations; seven of 15 and one of 15 animal room samples for rats and mice, respectively, were within 10% of the target concentrations.

DIET PREPARATION
- Rate of preparation of diet (frequency): five times
- Storage temperature of food: The dose formulations were stored in double polyethylene bags with twist-ties at room temperature for up to 48 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity studies of 0.03125% and 0.5% formulations and stability studies of 0.005% and 0.03125% formulations were performed by the analytical chemistry laboratory using GC. Additional homogeneity studies of the 0.025% and 0.4% dose formulations were performed by the study laboratory using GC. Homogeneity was confirmed, and stability was confirmed for at least 48 days for dose formulations stored in sealed plastic bags protected from light at room temperature and below, and for at least 7 days under simulated animal room conditions if the dosed feed was kept free from contamination with rodent urine and feces.
Periodic analyses of the dose formulations of methyl trans-styryl ketone were conducted by the study laboratory using GC. The dose formulations were analyzed three times; animal room samples of these dose formulations were also analyzed. Of the dose formulations analyzed, 15 of 17 for rats and mice were within 10% of the target concentrations; seven of 15 and one of 15 animal room samples for rats and mice, respectively, were within 10% of the target concentrations.

Duration of treatment / exposure:

14 weeks

Frequency of treatment:

ad libitum, available in diet for 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.025 other: %

Remarks:

approx. 18 and 19 mg/kg bw in females and males, respectively
Basis:
nominal in diet

Doses / concentrations 2

Dose / conc.:

0.05 other: %

Remarks:

approx. 36 and 38mg/kg bw in females and males, respectively
Basis:
nominal in diet

Doses / concentrations 3

Dose / conc.:

0.1 other: %

Remarks:

approx. 72 and 77 mg/kg bw in females and males, respectively
Basis:
nominal in diet

Doses / concentrations 4

Dose / conc.:

0.2 other: %

Remarks:

approx. 145 and 150 mg/kg bw in females and males, respectively
Basis:
nominal in diet

Doses / concentrations 5

Dose / conc.:

0.4 other: %

Remarks:

approx. 290 and 300 mg/kg bw in females and males,respectively
Basis:
nominal in diet

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes

Details on study design:

- Dose selection rationale: based on results of a dosed-feed palatability study.
- Rationale for animal assignment: Animals were distributed randomly into groups of approximately equal initial mean body weights.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: no data

BODY WEIGHT: Yes
- Time schedule for examinations: core study animals were weighed initially, weekly and at the end of the studies

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed consukmption was recorded weekly by cage.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 4 and 24 and from core study animals at the end of the studies for haematology and clinical chemistry (rats).
- Anaesthetic used for blood collection: Yes (70% CO2/30% O2 mixture)
- Animals fasted: No data
- How many animals:
- Parameters examined: haematocrit; haemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was collected for hematology analyses from surviving mice at study termination.
- Anaestethitc used for blood collection: Yes (70% CO2/30% O2 mixture)
- Animals fasted: No data
- How many animals:
- Parameters examined: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts

OTHER:
Type and Frequency of Observation
Observed twice daily; core study animals were weighed initially, weekly, and at the end of the studies; clinical findings were recorded initially, weekly, and at the end of the studies. Feed consumption was recorded weekly by cage.

Sperm Motility and Vaginal Cytology
At the end of the studies, spermatid and sperm samples were collected from male animals in the 0%, 0.1%, 0.2%, and 0.4% groups. The following parameters were evaluated: spermatid heads per testis and per gram testis, sperm motility, and sperm per cauda epididymis and per gram cauda epididymis. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 consecutive days prior to the end of the studies from females exposed to 0%, 0.1%, 0.2%, and 0.4%.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsies were performed on all core study animals. The heart, right kidney, liver, lung, right testis, and thymus were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin (except eyes were first fixed in Davidson’s solution), processed and trimmed, embedded in paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin. Complete histopathologic examinations were performed on 0% and 0.4% core study rats and mice; the kidney, nose, and stomach were examined in all exposed groups of core study rats and mice. After a review of the laboratory reports and selected histopathology slides by a quality assessment pathologist, the findings and reviewed slides were submitted to a NTP Pathology Working Group (PWG) coordinator for a second independent review.

HISTOPATHOLOGY: Yes
Complete histopathology was performed on 0% and 0.4% core study rats and mice. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, esophagus, eye, gallbladder (mice), Harderian gland, heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus. In addition, the kidney, nose, and stomach were examined in the remaining exposed groups.

Other examinations:

At the end of the 3-month feed studies, samples were collected for sperm motility and vaginal cytology evaluations on rats and mice exposed to 0%, 0.1%, 0.2%, or 0.4%. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females were moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus). Male animals were evaluated for sperm count and motility. The left testis and left epididymis were isolated and weighed. The tail of the epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed. Test yolk (rats) or modified Tyrode’s buffer (mice) was applied to slides and a small incision was made at the distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify spermatogenesis, the tes-ticular spermatid head count was determined by removing the tunica albuginea and homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenization-resistant spermatid nuclei were counted with a haemacytometer.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All core study rats survived to the end of the study. Clinical findings included diarrhea and hyperactivity in males and females.

Mortality:

mortality observed, treatment-related

Description (incidence):

All core study rats survived to the end of the study. Clinical findings included diarrhea and hyperactivity in males and females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Final mean body weights of 0.4% males and females and mean body weight gains of 0.4% males were significantly less than those of the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Feed consumption by exposed groups was similar to that by the controls.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

in male rats (0.4 %), the mean cell volume and the mean cell haemoglobin was significantly decreased on day a, and the platelets (at 0.2 and 0.4 %) were significantly increased on day 4.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

in males (0.4 %) urea nitrogen was sig. increased at day 24, creatinine (at 0.1, 0.2 and 0.4 %) was increased in week 14, and week 14 and day 4 and week 14, respectively. Moreover, Alanine aminotransferase was significantly elevated in males

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In all exposed males: treatment-related increased incidences of goblet cell hyperplasia of the resp. epithelium of the nose and nephropathy of the kidney. In females: increased incidence of goblet cell hyperplasia of the resp. epithelium (nose, 0.4 %).

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All core study rats survived to the end of the study. Clinical findings included diarrhea and hyperactivity in males and females.

BODY WEIGHT AND WEIGHT GAIN
Final mean body weights of 0.4% males and females and mean body weight gains of 0.4% males were significantly less than those of the controls.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Feed consumption by exposed groups was similar to that by the controls.

HAEMATOLOGY
In male rats, treated with 0.4 % in diet, the mean cell volume and the mean cell haemoglobin were significantly decreased on day 4,
Moreover, the platelets (at the dose levels 0.2 and 0.4 %) were significantly increased on day 4.
In female rats, the haematocrit and the haemoglobin were significantly (0.05 %) and the erythrocytes (0.01%) increased in animals treated with 0.4 % in diet at day 4.
On day 4, an increase in the erythron, evidenced by increases in the hematocrit, hemoglobin, and erythrocyte count values occurred in females exposed to 0.4%. The erythron increase was transient (occurred only on day 4) and minimal (≤10%) and would be consistent with a transient physiologic hemoconcentration possibly related to a transient decrease in water intake (dehydration) early in the study.

CLINICAL CHEMISTRY
In males treated with 0.4 %, the urea nitrogen was significantly increased at day 24,
In addition, creatinine (at the dose levels 0.1, 0.2 and 0.4 %) was increased in week 14, week 14 and day 4 and week 14, respectively.
Moreover, Alanine aminotransferase was significantly elevated in males (at the dose levels: 0.025, 0.05, 0.1, 0.2 and 0.4), mostly at the later timepoints (day 24 and week 14).
The alkaline phosphatase was significantly decreased in the dose levels 0.05, 0.1, 0.2 and 0.4 % (also at the later timepoints (day 24 and week 14).
In females, the alanine aminotransferase was significantly inceases in the dose group 0.4 % on day 4 ( P = 0.05) and in week 14 (p = 0.01).
IN addition, the bile salt were significantly increased in the dose group 0.4 % on day 4 (P = 0.05).
At week 14, serum chemistry evaluations demonstrated a small (up to 40%), treatment-related decrease in serum alanine aminotransferase (ALT) activity in all exposed male groups and the female group exposed to 0.4%; males exposed to 0.05% or 0.4% were also affected on day 24. The significance or mechanism for the decrease in serum ALT activity is unknown, and decreased activity has not been considered to be a pathologically important event (Hall, 2007). No other changes in the hematology or serum chemistry variables were considered attributable to methyl trans-styryl ketone exposure.

ORGAN WEIGHTS
The relative R Kidney weight was significantly inceased in males at 0.4 % (P = 0.05).
The relative liver weight was also significantly increased in males treated with 0.4 % (P = 0.01)
In females the absolute thymus weight was significantly decreased at the dose level 0.4 %.
No biologically significant organ weight changes were observed in exposed groups of males or females

HISTOPATHOLOGY: NON-NEOPLASTIC
In all exposed groups of males, there were treatment-related increased incidences of goblet cell hyperplasia of the respiratory epithelium of the nose and nephropathy of the kidney. In females, there was an increased incidence of goblet cell hyperplasia of the respiratory epithelium of the nose in the 0.4% group.
In the nose of male rats, there were treatment-related increased incidences of goblet cell hyperplasia of the respiratory epithelium in all exposed groups with slight increases in the severities of this lesion in the groups exposed to 0.1% and 0.4%. Goblet cell hyperplasia involved the respiratory epithelium lining the nasal septum and dorsal meatus in the Level I section and was characterized by increases in the size and numbers of goblet cells with pseudo-gland formation. A few of the pseudo-glands contained foci of necrotic cells forming clumps of pyknotic nuclear debris.
In the kidney of male rats, there were slight treatment-related increased incidences of nephropathy in all exposed groups with an increased severity in the group exposed to 0.4%. Nephropathy was characterized by necrosis and degeneration of scattered renal tubules, some with tubular regeneration. Regenerative tubules had increased numbers of cells with more intense basophilic staining and slightly thickened basement membranes. Minimal interstitial fibrosis with a few mononuclear cell aggregates was also noted.

OTHER FINDINGS
Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male rats; however, it exhibits potential for reproductive toxicity in female rats based upon an increased probability of extended diestrus at the highest exposure concentration.
There were no significant differences in any of the reproductive organ weights or sperm parameters of male rats at any exposure concentration. There were no changes in the proportion of regularly cycling females, estrous cycle length, or percentage of time spent in the individual stages of the estrous cycle of female rats at any exposure concentration; however, females exposed to 0.4% had a significantly higher probability of extended diestrus than the controls (P=0.035).

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

145 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 2 Survival, Body Weights, and Feed Consumption of Rats in the 3-Month Feed Study of Methyl trans-Styryl Ketone^a
Concentration(%)	Survival^b	Final Weight
		Initial Body Weight (g)	Final Body Weight (g)	Change in Body Weight (g)	Relative to Controls (%)	Feed Consumption Week1	Feed Consumption Week14
Male
0	10/10	103±2	335±7	233±6		16.6	17.2
0.025	10/10	101±3	349±7	248±6	104	16.9	17.9
0.05	10/10	102 ± 3	331 ± 8	229 ± 7	99	17.1	17.8
0.1	10/10	103 ± 3	330 ± 4	227 ± 5	99	16.5	17.8
0.2	10/10	101 ± 2	324 ± 3	223 ± 4	97	15.9	17.3
0.4	10/10	102 ± 3	317±5*	215 ± 3*	95	14.4	17.8
Female
0	10/10	93 ± 2	187 ± 3	94 ± 3		12.4	11.1
0.025	10/10	90 ± 2	183 ± 3	93 ± 3	98	12.5	11.1
0.05	10/10	94 ± 2	180 ± 2	86 ± 2	96	11.9	10.7
0.1	10/10	92 ± 2	181 ± 3	89 ± 2	97	12.2	10.7
0.2	10/10	92 ± 1	184 ± 4	92 ± 4	98	12.1	10.7
0.4	10/10	90 ± 2	175 ± 4*	85 ± 3	93	11.6	10.1
*  Significantly different (P<0.05) from the control group by Williams' or Dunnett's test
^a  Weights and weight changes are given as mean ± standard error. Feed consumption is expressed as grams per animal per day.
^b  Number of animals surviving at14weeks/number initially in group
Table 3 Incidences of Selected Nonneoplastic Lesions in Rats in the 3-Month Feed Study of Methyltrans-Styryl Ketone
	0%	0.025%	0.05%	0.1%	0.2%	0.4%
Male
Kidney^aNephropathy*^5	10 6 (1.0)^c	10 8 (1.0)	108 (1.0)	109 (1.0)	10 10* (1.0)	10 10* (1.6)
Nose Respiratory Epithelium, Hyperplasia, Goblet Cell	10 0	10 4* (1.0)^c	103 (1.0)	10 4* (1.3)	103 (1.0)	10 9** (1.3)
Female
Nose Respiratory Epithelium, Hyperplasia, Goblet Cell	10 3 (1.0)	10 0	103 (1.0)	103 (1.0)	103 (1.0)	10 7 (1.0)
*  Significantly different (P<0.05) from the control group by the Fisher exact test
** P<0.01
^a  Number of animals with tissue examined microscopically
^b  Number of animals with lesion
^c  Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Table 4 Haematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study of Methyl trans-Styryl Ketone^a
	0%	0.025%	0.05%	0.1%	0.2%	0.4%
Male
Hematology
n Day4	10	9	10	10	9	10
Day24	10	8	10	9	10	10
Week14	9	10	10	10	10	10
Hematocrit (%)
Day 4	41.6±1.0	41.2 ± 1.8	41.1 ± 0.9	41.7 ± 1.2	39.9 ± 0.6	41.6 ± 0.9
Day 24	43.2±0.3	43.0 ± 0.4	44.1 ± 0.3	43.8 ± 0.5	43.3 ± 0.7	43.8 ± 0.4
Week14	46.3 ± 0.3	45.9 ± 0.3	46.6 ± 0.2	45.6 ± 0.5	46.0 ± 0.4	44.9 ± 0.6
Hemoglobin (g/dL)
Day 4	14.3 ± 0.3	14.1 ± 0.6	14.1 ± 0.3	14.3 ± 0.4	13.7 ± 0.2	14.2 ± 0.3
Day 24	15.0±0.1	14.9 ± 0.1	15.2 ± 0.1	15.1 ± 0.2	15.0 ± 0.3	15.2 ± 0.1
Week 14	15.4±0.1	15.3 ± 0.1	15.5±0.1	15.2 ± 0.1	15.2 ± 0.1	15.0 ± 0.2
Erythrocytes (10^6/uL)
Day 4	7.48±0.18	7.42 ± 0.30	7.41 ± 0.15	7.55 ± 0.20	7.24 ± 0.10	7.57 ± 0.14
Day 24	7.77±0.06	7.79 ± 0.08	7.96 ± 0.05	7.89 ± 0.09	7.78 ± 0.13	7.86 ± 0.07
Week 14	8.82 ± 0.05	8.76 ± 0.06	8.77 ± 0.08	8.68 ± 0.08	8.70 ± 0.07	8.53 ± 0.12
Reticulocytes (10^6/uL)
Day 4	0.48 ± 0.02	0.50 ± 0.04	0.48 ± 0.02	0.54 ± 0.05	0.49 ± 0.03	0.51 ± 0.01
Day 24	0.28 ± 0.01	0.29 ± 0.01	0.29 ± 0.01	0.30 ± 0.01	0.29 ± 0.02	0.28 ± 0.02
Week 14	0.26±0.02	0.26 ± 0.01	0.30 ± 0.01	0.25 ± 0.02	0.27 ± 0.01	0.24 ± 0.02
Mean cell volume (fL)
Day 4	55.8±0.2	55.7 ± 0.2	55.3±0.2	55.2 ± 0.2	55.2 ± 0.2	55.0 ± 0.3*
Day 24	55.5 ± 0.3	55.1 ± 0.2	55.2 ± 0.2	55.3 ± 0.2	55.6 ± 0.3	55.7 ± 0.2
Week 14	52.6±0.2	52.2 ± 0.1	53.3±0.4	52.5 ± 0.2	52.8 ± 0.2	52.7 ± 0.2
Mean cell hemoglobin (pg)
Day 4	19.1 ± 0.1	19.0 ± 0.1	19.0 ± 0.1	18.9 ± 0.1	18.9 ± 0.1	18.8 ± 0.1*
Day 24	19.3 ± 0.1	19.2 ± 0.1	19.1 ± 0.1	19.2 ± 0.1	19.3 ± 0.1	19.3 ± 0.1
Week 14	17.5 ± 0.1	17.4 ± 0.0	17.7 ± 0.1	17.5 ± 0.0	17.5 ± 0.1	17.6 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4	34.2 ± 0.1	34.2 ± 0.1	34.2 ± 0.1	34.3 ± 0.1	34.3 ± 0.1	34.1 ± 0.1
Day 24	34.7 ± 0.1	34.7 ± 0.1	34.6 ± 0.1	34.6 ± 0.1	34.7 ± 0.1	34.6 ± 0.0
Week 14	33.3 ± 0.1	33.3 ± 0.1	33.3 ± 0.1	33.4 ± 0.1	33.1 ± 0.1	33.5 ± 0.1
Platelets (10^3/uL)
Day 4	646.2 ± 16.6	709.7 ± 15.8	678.3 ± 22.1	689.0 ± 25.1	736.9±13.1**	734.1 ± 26.3**
Day 24	612.7 ± 17.6	636.9 ± 7.6	652.0 ± 11.5	649.3 ± 17.2	625.8 ± 12.6	656.6 ± 10.7
Week 14	502.3 ± 14.9	501.2 ± 13.5	490.6 ± 10.4	502.2 ± 13.9	508.6 ± 14.5	492.3 ± 17.5
Leukocytes (10^3/uL)
Day 4	9.42 ± 0.56	8.97 ± 0.69	9.07 ± 0.62	8.74 ± 0.62	8.80 ± 0.41	8.33 ± 0.29
Day 24	10.61 ± 0.31	10.49 ± 0.46	10.46 ± 0.60	10.90 ± 0.44	10.56 ± 0.33	10.86 ± 0.34
Week 14	9.74 ± 1.36	13.72 ± 1.55	14.19 ± 2.33	9.91 ± 1.70	12.17 ± 1.38	11.95 ± 1.39
Segmented neutrophils (10^3/uL)
Day 4	1.22 ± 0.11	1.20 ± 0.15	1.34 ± 0.11	1.05±0.12	1.16 ± 0.07	1.05± 0.10^b
Day 24	0.82 ± 0.08	0.82 ± 0.11	0.96 ± 0.12	1.11 ± 0.12	0.86 ± 0.12	0.81 ± 0.11
Week 14	1.22 ± 0.19	1.56 ± 0.25	1.72 ± 0.24	1.26 ± 0.26	1.43 ± 0.19	1.23 ± 0.15
Lymphocytes (10^3/uL)
Day 4	7.24 ± 0.58	6.67 ± 0.50	6.72 ± 0.55	6.74 ± 0.45	6.64 ± 0.42	6.11 ± 0.25^b
Day 24	8.98 ± 0.24	8.99 ± 0.42	8.77 ± 0.46	8.92 ± 0.38	8.84 ± 0.21	9.37 ± 0.32
Week 14	7.86±1.09	11.45 ± 1.35	11.43 ± 1.96	7.82 ± 1.27	10.00 ± 1.19	9.99 ± 1.21
Monocytes (10^3/uL)
Day 4	0.88 ± 0.08	1.06 ± 0.17	0.96 ± 0.16	0.90 ± 0.15	0.89 ± 0.13	1.08 ± 0.22^b
Day 24	0.77 ± 0.09	0.61 ± 0.12	0.70 ± 0.09	0.81 ± 0.10	0.83 ± 0.12	0.65 ± 0.16
Week 14	0.62 ± 0.12	0.68 ± 0.06	0.89 ± 0.14	0.77 ± 0.18	0.65 ± 0.14	0.64 ± 0.09
Basophils (10^3/uL)
Day 4	0.000±0.000	0.000±0.000	0.000±0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000^b
Day 24	0.000±0.000	0.000±0.000	0.000±0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000
Week 14	0.000±0.000	0.000±0.000	0.000±0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000
Eosinophils (10^3/uL)
Day 4	0.05 ± 0.02	0.02 ± 0.01	0.06±0.02	0.02 ± 0.02	0.04 ± 0.02	0.03 ± 0.01^b
Day 24	0.03 ± 0.02	0.03 ± 0.03	0.02 ± 0.01	0.02 ± 0.02	0.02 ± 0.01	0.00 ± 0.00
Week 14	0.04 ± 0.03	0.02 ± 0.02	0.14 ± 0.05	0.03 ± 0.03	0.07 ± 0.02	0.04 ± 0.03
Clinical Chemistry
Day 4	10	10	10	10	10	10
Day 24	10	8	10	9	10	10
Week 14	10	10	10	10	10	10
Urea nitrogen (mg/dL)
Day 4	14.1 ± 0.5	13.9 ± 0.5	14.2 ± 0.4	14.7 ± 0.4	13.4 ± 0.6	14.7 ± 0.8
Day 24	13.5±0.5	12.5 ± 0.6	15.9 ± 0.5	14.7 ± 0.4	13.8±0.5	18.0 ± 0.6**
Week 14	16.3±0.5	17.1 ± 0.5	16.9 ± 0.5	16.0 ± 0.4	17.2 ± 0.6	18.5±0.9
Creatinine (mg/dL)
Day 4	0.31 ± 0.02	0.31 ± 0.01	0.33±0.02	0.35 ± 0.02	0.34 ± 0.02	0.41 ± 0.02**
Day 24	0.39 ± 0.01	0.39 ± 0.01	0.39 ± 0.02	0.39 ± 0.01	0.41 ± 0.01	0.42 ± 0.01
Week 14	0.46±0.02	0.51 ± 0.02	0.49 ± 0.02	0.51 ± 0.01*	0.57 ± 0.02**	0.58 ± 0.03**
Total protein (g/dL)
Day 4	5.9 ± 0.1	5.9 ± 0.1	5.9 ± 0.1	6.0 ± 0.1	5.9 ± 0.1	6.1 ± 0.1
Day 24	6.8 ± 0.1	6.7 ± 0.1	6.6±0.0	6.8 ± 0.1	6.9 ± 0.1	6.9 ± 0.1
Week 14	7.4 ± 0.1	7.3±0.1	7.4 ± 0.1	7.5 ± 0.1	7.5 ± 0.1	7.5 ± 0.1
Albumin (g/dL)
Day 4	4.0±0.0	4.0±0.1	4.0±0.1	4.1 ± 0.1	4.0 ± 0.0	4.1 ± 0.0
Day 24	4.5 ± 0.0	4.5 ± 0.1	4.4 ± 0.0*	4.5 ± 0.0	4.5 ± 0.0	4.5 ± 0.0
Week 14	4.7 ± 0.0	4.6±0.0	4.8 ± 0.1	4.8 ± 0.1	4.8 ± 0.0	4.8 ± 0.1
Globulin (g/dL)
Day 4	1.9 ± 0.0	1.9 ± 0.1	1.9 ± 0.1	2.0 ± 0.0	1.9 ± 0.0	2.0 ± 0.0
Day 24	2.3 ± 0.0	2.2 ± 0.0	2.3 ± 0.0	2.3 ± 0.0	2.3 ± 0.0	2.4 ± 0.0
Week 14	2.7 ± 0.0	2.7 ± 0.1	2.6±0.1	2.7 ± 0.0	2.7 ± 0.0	2.7 ± 0.0
Albumin/globulin ratio
Day 4	2.1 ± 0.0	2.2 ± 0.1	2.2 ± 0.0	2.1 ± 0.0	2.1 ± 0.0	2.1 ± 0.0
Day 24	2.0±0.0	2.0 ± 0.0	2.0±0.0	2.0 ± 0.0	2.0 ± 0.0	1.9 ± 0.0
Week 14	1.8±0.0	1.7 ± 0.0	1.8±0.0	1.8±0.0	1.8 ± 0.0	1.8 ± 0.0
Alanine aminotransferase (IU/L)
Day 4	60 ± 2	65 ± 2	60 ± 2	62 ± 2	61 ± 2	62 ± 1
Day 24	43 ± 1	42 ± 1	37 ± 0**	39 ± 1	44 ± 1	37 ± 1**
Week 14	86 ± 5	71 ± 4*	65 ± 6**	69 ± 4*	57 ± 4**	56 ± 3**
Alkaline phosphatase (IU/L)
Day 4	837 ± 21	819 ± 24	815±19	841 ± 17	798 ± 19	767 ± 12
Day 24	542 ± 7	531 ± 9	508 ± 7**	511 ± 9*	500 ± 7**	489 ± 10**
Week 14	238 ± 4	239 ± 4	233 ± 7	239 ± 4	229 ± 4	233 ± 5
Creatine kinase (IU/L)
Day 4	452 ± 62	590±82	625 ± 91	577 ± 52	459 ± 25	495 ± 51
Day 24	298 ± 60	266 ± 33	263 ± 35	370 ± 88	309 ± 32	236 ± 19
Week 14	222 ± 36	220 ± 27	228 ± 31	240 ± 33	198 ± 16	236 ± 34
Sorbitol dehydrogenase (IU/L)
Day 4	14 ± 1	13±1	15±1	12 ± 1	16 ± 1	13 ± 1
Day 24	20 ± 1	20 ± 2	19 ± 1	18 ± 2	20 ± 1	19 ± 1
Week 14	30 ± 2	30 ± 1	29 ± 2	30 ± 1	28 ± 1	26 ± 1
Bile salts (umol/L)
Day 4	25.7 ± 1.9	28.0 ± 2.6	36.1 ± 2.0**	28.6 ± 1.9	32.5 ± 1.3	35.0 ± 3.0
Day 24	24.7 ± 2.9	27.9 ± 2.1	25.7 ± 2.7	24.4 ± 2.8	28.4 ± 2.0	20.3 ± 1.3
Week 14	21.9 ± 2.3	21.3 ± 1.9	19.1 ± 1.4	24.5 ± 1.3	25.8 ± 2.8	24.6 ± 1.9
Female
Hematology
Day 4	10	10	9	10	10	10
Day 24	10	10	10	10	10	9
Week 14	10	10	10	10	10	10
Hematocrit (%)
Day 4	40.8±0.5	41.2 ± 0.4	41.7 ± 0.6	40.2 ± 0.4	42.2 ± 0.8	44.3 ± 1.9*
Day 24	45.8±0.6	45.7 ± 0.4	45.8±0.4	44.9 ± 0.5	45.5 ± 0.4	45.5 ± 0.4
Week 14	43.1 ± 0.6	43.0 ± 0.3	42.9 ± 0.4	43.1 ± 0.3	43.1 ± 0.3	43.2 ± 0.5
Hemoglobin (g/dL)
Day 4	14.0 ± 0.2	14.2 ± 0.1	14.3 ± 0.2	13.9 ± 0.1	14.5 ± 0.2	15.3 ± 0.6*
Day 24	16.2 ± 0.2	16.2 ± 0.1	16.2 ± 0.2	15.8 ± 0.2	16.0 ± 0.1	16.1 ± 0.1
Week 14	14.9 ± 0.1	14.8 ± 0.1	14.7 ± 0.1	14.9 ± 0.1	14.8 ± 0.1	14.8 ± 0.2
Erythrocytes (10^6/uL)
Day 4	7.41 ± 0.08	7.52 ± 0.08	7.63 ± 0.10	7.34 ± 0.07	7.74 ± 0.14	8.16 ± 0.42**
Day 24	8.35 ± 0.11	8.36 ± 0.09	8.33 ± 0.07	8.16 ± 0.08	8.27 ± 0.09	8.29 ± 0.06
Week 14	7.89 ± 0.11	7.90 ± 0.05	7.84 ± 0.06	7.92 ± 0.06	7.88 ± 0.06	7.94 ± 0.09
Reticulocytes (10^6/uL)
Day 4	0.34 ± 0.02	0.34 ± 0.02	0.34 ± 0.02	0.33 ± 0.02	0.32 ± 0.02	0.33 ± 0.03
Day 24	0.22 ± 0.02	0.23 ± 0.02	0.23 ± 0.02	0.22 ± 0.03	0.22 ± 0.02	0.22 ± 0.03
Week 14	0.25 ± 0.01	0.27 ± 0.02	0.29 ± 0.02	0.25 ± 0.01	0.28 ± 0.02	0.27 ± 0.02
Mean cell volume (fL)
Day 4	55.0±0.2	54.8 ± 0.2	54.8±0.2	54.9 ± 0.2	54.5 ± 0.2	54.6 ± 0.2
Day 24	54.9 ± 0.2	54.7 ± 0.3	54.9 ± 0.2	55.1 ± 0.2	55.0 ± 0.3	54.9 ± 0.3
Week 14	54.7 ± 0.2	54.3 ± 0.2	54.9 ± 0.1	54.5 ± 0.2	54.8 ± 0.2	54.4 ± 0.2
Mean cell hemoglobin (pg)
Day 4	19.0 ± 0.1	18.9 ± 0.1	18.8±0.1	19.0 ± 0.1	18.7 ± 0.1	18.9 ± 0.1
Day 24	19.4 ± 0.1	19.4 ± 0.1	19.4 ± 0.1	19.4 ± 0.0	19.4 ± 0.1	19.5 ± 0.1
Week 14	18.8±0.1	18.7 ± 0.1	18.8±0.1	18.8 ± 0.1	18.9 ± 0.1	18.7 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4	34.4 ± 0.1	34.5 ± 0.1	34.4 ± 0.1	34.6 ± 0.1	34.4 ± 0.1	34.6 ± 0.1
Day24	35.4±0.1	35.5 ± 0.1	35.3±0.1	35.3 ± 0.1	35.3 ± 0.1	35.4 ± 0.1
Week 14	34.5 ± 0.2	34.3 ± 0.1	34.3±0.1	34.6 ± 0.1	34.4 ± 0.1	34.4 ± 0.1
Platelets (10^3/u.T.)
Day 4	739.6±14.9	696.4 ± 21.3	737.8 ± 32.7	678.9 ± 31.5	681.3 ± 25.4	702.3 ± 43.3
Day 24	572.0±27.6	588.4 ± 12.7	557.1 ± 13.6	596.4 ± 13.4	581.4 ± 12.5	588.7 ± 12.8
Week 14	545.4 ± 18.8	549.0±19.2	539.6 ± 19.7	567.0 ± 21.9	507.8 ± 19.6	556.9 ± 18.9
Leukocytes (10^3/uL)
Day 4	10.62 ± 0.31	10.65 ± 0.31	10.93 ± 0.35	10.70 ± 0.42	10.15±0.59	9.83 ± 0.77
Day 24	10.13±0.46	10.50 ± 0.54	10.51 ± 0.41	10.50 ± 0.58	10.36 ± 0.78	10.93 ± 0.50
Week 14	9.29 ± 1.73^b	10.83±1.31	8.35 ± 1.46	9.52 ± 1.66	10.25±1.79	8.59 ± 1.00
Segmented neutrophils (10^3/uL)
Day 4	1.83±0.14	1.90 ± 0.12	2.17 ± 0.11	1.92 ± 0.16	1.70 ± 0.14	1.80 ± 0.17
Day 24	0.95 ± 0.07	0.97 ± 0.07	0.96 ± 0.10	0.79 ± 0.08	1.13 ± 0.13	1.11 ± 0.12
Week 14	1.02 ± 0.23^b	1.06 ± 0.15	0.97 ± 0.15	1.09 ± 0.29	0.92 ± 0.19	0.80 ± 0.10
Lymphocytes (10^3/uL)
Day 4	8.34 ± 0.34	8.18±0.31	8.17 ± 0.38	8.07 ± 0.26	8.05 ± 0.55	7.51 ± 0.60
Day 24	8.60±0.43	8.94 ± 0.48	8.89 ± 0.33	9.13±0.46	8.61 ± 0.65	9.07 ± 0.50
Week 14	7.79 ± 1.40^b	9.34 ± 1.19	6.93 ± 1.25	7.98 ± 1.32	8.98 ± 1.55	7.39 ± 0.93
Monocytes (10^3/uL)
Day 4	0.42 ± 0.03	0.51 ± 0.05	0.55 ± 0.09	0.63 ± 0.09	0.38 ± 0.11	0.44 ± 0.08
Day 24	0.49 ± 0.06	0.54 ± 0.10	0.59 ± 0.08	0.51 ± 0.10	0.56 ± 0.10	0.63 ± 0.07
Week 14	0.35 ± 0.07^b	0.42 ± 0.07	0.36 ± 0.07	0.32 ± 0.05	0.28 ± 0.06	0.32 ± 0.05
Basophils (10^3/uL)
Day 4	0.000±0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000
Day 24	0.000±0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000
Week 14	0.000± 0.000^b	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000	0.000 ± 0.000
Eosinophils (10^3/uL)
Day 4	0.03 ± 0.02	0.06 ± 0.03	0.04 ± 0.03	0.08 ± 0.03	0.02 ± 0.02	0.09 ± 0.04
Day 24	0.08 ± 0.03	0.05 ± 0.02	0.07 ± 0.02	0.06 ± 0.02	0.07 ± 0.02	0.09 ± 0.03
Week 14	0.09 ± 0.05^b	0.02 ± 0.01	0.07 ± 0.03	0.10 ± 0.02	0.04 ± 0.02	0.05 ± 0.02
Clinical Chemistry
Day 4	10	10	10	10	10	9
Day 24	10	10	10	10	10	9
Week 14	10	10	10	10	10	10
Urea nitrogen (mg/dL)
Day 4	12.5 ± 0.5	14.0 ± 0.6	12.9 ± 0.4	12.8±0.6	13.1 ± 0.4	14.7 ± 0.7
Day 24	16.9 ± 0.5	16.0 ± 0.7	17.6 ± 0.5	16.7 ± 0.5	15.4 ± 0.5	19.1 ± 0.7
Week 14	17.5 ± 0.3	17.2 ± 0.5	17.3 ± 0.4	17.1 ± 0.3	16.6 ± 0.5	17.0 ± 0.4
Creatinine (mg/dL)
Day 4	0.42 ± 0.01	0.39 ± 0.02	0.40 ± 0.01	0.41 ± 0.02	0.42 ± 0.01	0.47 ± 0.02
Day 24	0.41 ± 0.01	0.40 ± 0.00	0.40 ± 0.01	0.41 ± 0.01	0.41 ± 0.01	0.39 ± 0.02
Week 14	0.56±0.02	0.55 ± 0.02	0.58 ± 0.02	0.53 ± 0.02	0.57 ± 0.02	0.56 ± 0.02
Total protein (g/dL)
Day 4	6.2 ± 0.1	6.1 ± 0.1	6.1 ± 0.1	6.2 ± 0.1	6.2 ± 0.1	6.2 ± 0.1
Day 24	6.7 ± 0.1	6.5 ± 0.1	6.5 ± 0.1	6.7 ± 0.1	6.6 ± 0.0	6.6 ± 0.1
Week 14	7.2 ± 0.1	7.3 ± 0.1	7.2 ± 0.1	7.2 ± 0.1	7.3 ± 0.1	7.3 ± 0.1
Albumin (g/dL)
Day 4	4.4 ± 0.0	4.3 ± 0.0	4.3 ± 0.0	4.3 ± 0.0	4.3 ± 0.0	4.3 ± 0.1
Day 24	4.6±0.0	4.6 ± 0.0	4.5 ± 0.0	4.6 ± 0.0	4.5 ± 0.1	4.6 ± 0.1
Week 14	5.0±0.1	5.1 ± 0.1	5.0 ± 0.1	5.0 ± 0.1	5.0 ± 0.1	5.0 ± 0.1
Globulin (g/dL)
Day 4	1.8±0.1	1.8 ± 0.0	1.8 ± 0.1	1.8 ± 0.0	1.9 ± 0.1	1.9 ± 0.1
Day 24	2.1 ± 0.0	2.0 ± 0.0	2.0 ± 0.1	2.0 ± 0.0	2.1 ± 0.1	2.0 ± 0.0
Week 14	2.2 ± 0.1	2.2 ± 0.1	2.2 ± 0.1	2.2 ± 0.1	2.3 ± 0.1	2.2 ± 0.1
Albumin/globulin ratio
Day 4	2.4 ± 0.1	2.4 ± 0.1	2.4 ± 0.1	2.4 ± 0.1	2.3 ± 0.1	2.3 ± 0.1
Day 24	2.2 ± 0.0	2.3 ± 0.0	2.2 ± 0.1	2.3 ± 0.0	2.2 ± 0.1	2.2 ± 0.0
Week 14	2.3 ± 0.1	2.3 ± 0.1	2.3 ± 0.1	2.3 ± 0.1	2.2 ± 0.1	2.2 ± 0.0
Alanine aminotransferase (IU/L)
Day 4	47 ± 1	50 ± 1	48 ± 1	49 ± 1	50 ± 2	52 ± 1*
Day 24	35 ± 1	34 ± 1	32 ± 1	32 ± 1	35 ± 1	32 ± 1
Week 14	85 ± 10	67 ± 5	69 ± 10	61 ± 5	68 ± 8	49 ± 3**
Alkaline phosphatase (IU/L)
Day 4	583 ± 13	565 ± 16	580 ± 11	582 ± 15	580 ± 19	587 ± 11
Day 24	370 ± 9	357 ± 7	355 ± 6	349 ± 11	374 ± 7	353 ± 3
Week 14	180±8	190 ± 5	184 ± 4	189 ± 3	182 ± 3	182 ± 6
Creatine kinase (IU/L)
Day 4	423 ± 59	435 ± 63	395 ± 58	420 ± 79	488 ± 88	593 ± 115
Day 24	258 ± 32	295 ± 36	280 ± 30	304 ± 47	332 ± 27	307 ± 26
Week 14	278 ± 39	200 ± 21	231 ± 34	199 ± 25	182 ± 13	199 ± 15
Sorbitol dehydrogenase (IU/L)
Day 4	15±1	15 ± 1	18 ± 2	13 ± 1	12 ± 1	14 ± 1
Day 24	22 ± 1	19 ± 1	21 ± 2	22 ± 2	20 ± 2	19 ± 1
Week 14	31 ± 2	31 ± 2	32 ± 1	32 ± 1	33 ± 2	29 ± 1
Bile salts (umol/L)
Day 4	15.9 ± 1.3	22.9 ± 2.0*	23.2 ± 1.3*	23.7 ± 2.0*	20.6 ± 2.2	23.5 ± 2.4*
Day 24	17.7 ± 1.5	20.9 ± 1.9	22.0 ± 2.4	22.9 ± 1.5	18.7 ± 1.8	19.3 ± 2.4
Week 14	36.7 ± 4.0	39.1 ± 3.1	28.6 ± 1.0	45.7 ± 3.1	30.4 ± 2.8	29.7 ± 3.4
*  Significantly different (P<0.05) from the control group by Dunn's or Shirley's test
**P<0.01
^a  Data are presented as mean ± standard error. Statistical tests were performed on unrounded data.
^bn=9

Table 5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Feed Study of Methyltrans-Styryl Ketone^a
	0%	0.025%	0.05%	0.1%	0.2%	0.4%
Male
n	10	10	10	10	10	10
Necropsy bodywt	335 ± 7	349 ± 7	331 ± 8	330 ± 4	324 ± 3	317±5*
Heart
Absolute	0.92 ± 0.02	0.95 ± 0.02	0.89 ± 0.02	0.92 ± 0.01	0.90 ± 0.02	0.88 ± 0.02
Relative	2.741 ± 0.034	2.713 ± 0.023	2.697 ± 0.042	2.771 ± 0.021	2.787 ± 0.052	2.774 ± 0.038
R.Kidney
Absolute	1.01±0.02	1.02±0.03	0.99 ± 0.03	1.03±0.01	0.98 ± 0.02	1.01±0.02
Relative	3.010±0.046	2.937 ± 0.040	2.985 ± 0.024	3.115±0.051	3.029 ± 0.060	3.173±0.051*
Liver
Absolute	10.59±0.27	11.57±0.38	10.90 ± 0.54	10.66±0.23	10.59±0.15	11.01 ± 0.36
Relative	31.571 ± 0.313	33.138 ± 0.554	32.820 ± 0.981	32.274 ± 0.531	32.650 ± 0.328	34.675 ± 0.685**
Lung
Absolute	1.46±0.05	1.61 ± 0.09^b	1.39 ± 0.07	1.38±0.04	1.41 ± 0.04^b	1.44 ± 0.06
Relative	4.354 ± 0.129	4.600 ± 0.243^b	4.196±0.198	4.184±0.099	4.331 ± 0.146^b	4.534 ± 0.182
Thymus
Absolute	0.273 ± 0.010	0.285 ± 0.012	0.274 ± 0.009	0.272 ± 0.005	0.274 ± 0.007	0.242 ± 0.008
Relative	0.813±0.018	0.821 ± 0.039	0.832 ± 0.030	0.823 ± 0.012	0.846 ± 0.022	0.765 ± 0.027
Female
n	9	10	10	10	10	10
Necropsy body wt	187± 3^c	183 ± 3	180±2	181±3	184±4	175 ± 4*
Heart
Absolute	0.61 ± 0.01	0.60 ± 0.01	0.61 ± 0.01	0.61 ± 0.02	0.58 ± 0.01	0.58 ± 0.01
Relative	3.262 ± 0.055	3.276 ± 0.035	3.399 ± 0.050	3.372 ± 0.053	3.143±0.064	3.301 ± 0.028
R. Kidney
Absolute	0.64 ± 0.01	0.66 ± 0.01	0.64 ± 0.02	0.63 ± 0.01	0.65 ± 0.02	0.64 ± 0.02
Relative	3.428 ± 0.057	3.578 ± 0.054	3.541 ± 0.081	3.476 ± 0.046	3.504 ± 0.068	3.665 ± 0.093
Liver
Absolute	5.88 ± 0.12	5.79 ± 0.27	5.86 ± 0.19	5.69 ± 0.17	6.01 ± 0.15	5.92 ± 0.28
Relative	31.476 ± 0.383	31.451 ± 0.966	32.625 ± 0.899	31.478±0.564	32.690 ± 0.784	33.768 ± 1.018
Lung
Absolute	1.01 ± 0.06	0.93 ± 0.02	0.95 ± 0.05	0.96 ± 0.03	0.93 ± 0.02	0.92 ± 0.05
Relative	5.433 ± 0.298	5.070 ± 0.109	5.273 ± 0.268	5.301 ± 0.151	5.070 ± 0.145	5.238 ± 0.208
Thymus
Absolute	0.231 ± 0.007	0.225 ± 0.007	0.213±0.007	0.217±0.004	0.214±0.005	0.204 ± 0.005**
Relative	1.239 ± 0.030	1.224 ± 0.033	1.186±0.037	1.204 ± 0.021	1.164 ± 0.026	1.168±0.030
*  Significantly different (P<0.05) from the control group by Williams' or Dunnett's test
** Significantly different (P<0.01) from the control group by Williams' test
^a  Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).
^bn=9
^cn=10

Table 6 Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Feed Study of Methyltrans-Styryl Ketone^a
	0%	0.1%	0.2%	0.4%
n	10	10	10	10
Necropsy body wt	335 ± 7	330 ± 4	324 ± 3	317±5*
L. Cauda epididymis	0.1668±0.0039	0.1726±0.0028	0.1653 ± 0.0041	0.1637±0.0056
L. Epididymis	0.4611 ± 0.0048	0.4697 ± 0.0093	0.4664 ± 0.0105	0.4504 ± 0.0105
L. Testis	1.5126±0.0355	1.4703 ± 0.0187	1.5065 ± 0.0151	1.5014 ± 0.0174
Spermatid measurements
Spermatid heads (10^6/testis)	179.63 ± 7.44	179.06 ± 9.84	177.50 ± 5.51	190.50 ± 7.21
Spermatid heads (10^3/mg testis)	126.2 ± 3.4	130.8 ± 6.4	125.5 ± 4.0	136.3 ± 5.3
Epididymalspermatozoal measurements
Sperm motility (%)	77.7 ± 1.6	79.7 ± 1.1	81.0±0.7	72.4±8.3
Sperm (10^6/cauda epididymis)	82.8 ± 5.8	66.9 ± 9.6	59.6 ± 9.2	62.2±8.6
Sperm (10^3/mg cauda epididymis)	493 ± 25	402 ± 47	402 ± 38	397 ± 45
*  Significantly different (P<0.05) from the control group by Williams' test
^a  Data are presented as mean ± standard error. Differences from the control group are not significant by Dunnett's test (tissue weights) or Dunn's test (spermatid and epididymal spermatozoal measurements).
Table 7 Estrous Cycle Characterization for Female Rats in the 3-Month Feed Study of Methyltrans-Styryl Ketone^a
	0%	0.1%	0.2%	0.4%
Number weighed at necropsy	10	10	10	10
Necropsy body wt (g)	187±3	181 ± 3	184±4	175±4*
Proportion of regular cycling females^b	9/10	8/10	9/10	10/10
Estrous cycle length (days)	5.0 ± 0.16	5.1±0.12	5.0 ± 0.15	4.9 ± 0.07
Estrous stages(%of cycle)
Diestrus	63.3	60.8	60.0	57.5
Proestrus	7.5	13.3	11.7	10.8
Estrus	21.7	20.8	21.7	27.5
Metestrus	6.7	5.0	6.7	4.2
Uncertain diagnosis	0.8	0.0	0.0	0.0
*  Significantly different (P<0.05) from the control group by Dunnett's test
^a  Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the control group are not significant by Dunn's test (estrous cycle length). By multivariate analysis of variance, exposed females do not differ significantly from the control females in the relative length of time spent in the estrous stages. The tests for equality of transition probability matrices among all groups and between the control group and each exposed group indicated that female rats in the highest exposure group (0.4%) had a significantly higher probability of extended diestrus than controls (P=0.035).
^b  Number of females with a regular cycle/number of females cycling

Applicant's summary and conclusion

Conclusions:

The study was performed with the substance methyl trans-styryl ketone, equivalent or similar to OECD TG408 and therefore considered to be of high quality (reliability Klimisch 2). The validity criteria of the test system are fulfilled. The test material did not induce mortality and treatment-related clinical signs were diarrhoea and hyperactivity. An NOAEL was derived (145 (females, and 150 (males) mg/kg bw).

Executive summary:

The substance methyl trans-styryl ketone was investigated for its repeated dose toxicity via the oral route (NTP, 2011). Groups of 10 male and 10 female rats were fed diets containing 0%, 0.025%, 0.05%, 0.1%, 0.2%, or 0.4% methyl trans-styryl ketone (equivalent to average daily doses of approximately 18, 36, 72, 145, or 290 mg methyl trans-styryl ketone/kg body weight to males and 19, 38, 77, 150, or 300 mg/kg to females) for 14 weeks. Groups of 10 male and 10 female clinical pathology rats were fed the same concentrations for 24 days. All core study rats survived to the end of the study. Final mean body weights of males and females receiving 0.4% and mean body weight gains of males receiving 0.4% were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Clinical findings included diarrhoea and hyperactivity in males and females. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male rats; however, it exhibits potential for reproductive toxicity in female rats based upon an increased probability of extended diestrus at the highest exposure concentration. In all exposed groups of males, there were treatment-related increased incidences of goblet cell hyperplasia of the respiratory epithelium of the nose and nephropathy of the kidney. In females, there was an increased incidence of goblet cell hyperplasia of the respiratory epithelium of the nose in the group receiving 0.4%. An NOAEL was derived (145 (females), and 150 (males) mg/kg bw).