4-phenylbutenone

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

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 411 (Subchronic Dermal Toxicity: 90-Day Study)

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 reanalyses 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 to 5 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 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

Type of coverage:

not specified

Vehicle:

ethanol

Details on exposure:

TEST SITE
- Area of exposure: a shaved dorsal area posterior to the scapulae to the base of the tail.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5 (rats) or 2.0 (mice) mL/kg

VEHICLE
- Justification for use and choice of vehicle (if other than water): ethanol was used, due to the limited solubility of the test item in water
For the 3-month dermal studies, 95% ethanol was obtained as a single lot (R8092) from Pharmco Products, Inc. (Brookfield, CT), for use as the vehicle. Identity and purity analyses were conducted by the study laboratory. The chemical, a clear liquid, was identified as ethanol by infrared spectroscopy; the sample spectrum was essentially identical to a reference spectrum provided to the National Toxicology Program via Midwest Research Institute (Kansas City, MO). The purity of lot R8092 was determined by GC; no impurity peaks with areas exceeding 0.1% of the single major peak area were detected.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were prepared three times during the 3-month studies and approximately every 4 weeks during the 2-year studies by mixing methyl trans-styryl ketone and 95% ethanol to give the required concentrations. The dose formulations for the 3-month studies were stored at refrigerator temperatures under a headspace of inert gas in sealed amber glass vials for up to 42 days. The dose formulations for the 2-year studies were stored at room temperature in sealed amber glass containers for up to 42 days. A stability study of a 50 mg/mL formulation was performed by the analytical chemistry laboratory with GC. Stability was confirmed for at least 42 days for dose formulations stored in sealed amber glass containers at room temperature or below and for at least 3 hours under simulated animal room conditions if the dose containers were kept sealed except during the brief periods of removal of simulated doses. Additional stability studies of 5 and 180 mg/mL formulations were performed by Southern Research Institute using GC, and stability of dose formulations at these concentrations was confirmed for at least 42 days when stored refrigerated in sealed glass containers protected from light.
Periodic analyses of the dose formulations of methyl trans-styryl ketone were conducted by the study laboratories using GC. During the 3-month studies, the dose formulations were analyzed twice; animal room samples of these dose formulations were also analyzed. All 10 formulations for rats and mice were within 10% of the target concentrations; nine of 10 and six of eight animal room samples for rats and mice, respectively, were within 10% of the target concentrations. During the 2-year studies, the dose formulations were analyzed approximately every 2 months; animal room samples were also analyzed. All 33 formulations analyzed for rats and all 33 analyzed for mice were within 10% of the target concentrations; eight of 15 and nine of 15 animal room samples for rats and mice, respectively, were within 10% of the target concentrations. Evaporation of the ethanol vehicle during the dosing period is thought to be the reason for high animal room sample analysis results.

Duration of treatment / exposure:

5 days per week for 14 weeks

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

22 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

44 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

87.5 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

175 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

350 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/ sex / dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected using the limit of solubility of methyl trans-styryl ketone as the basis for the highest dose.
- 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: Observed twice daily, clinical findings were recorded weekly and at the end of the studies.

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION (if dermal study): Yes
- Time schdule: Observed twice daily, clinical findings were recorded weekly and at the end of the studies.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on days 4 and 24 and from core study rats and mice 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: hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:von days 4 and 24 and from core study rats and mice at the end of the studies for haematology and clinical chemistry (rats).
- 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:
Clinical findings were recorded weekly and at study termination for core study rats and mice. Core study animals were weighed initially, weekly, and at the end of the studies.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Necropsies were performed on all core study rats and mice. The heart, right kidney, liver, lung, right testis, and thymus were weighed.
HISTOPATHOLOGY: Yes
Complete histopathology was performed on core study 0 mg/kg rats and mice, 350 mg/kg rats, and 350, 700 and 1,400 mg/kg 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 (site of application), spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicle, thymus, thyroid gland, tongue, trachea, urinary bladder, and uterus. In addition, the adrenal gland (female mice), kidney, nose, skin, stomach, and uterus were examined in the remaining dosed groups.
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. The adrenal gland (female mice), kidney, nose, skin, stomach, and uterus were examined in all core study rats and mice.

Other examinations:

At the end of the 3-month studies, samples were collected from male rats and mice in the 0, 87.5, 175 and 350 mg/kg groups for sperm motility and vaginal cytology evaluations using the methods described for the 3-month feed studies.
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 in the 0, 87.5, 175, and 350 mg/kg groups.

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. Treatment-related clinical findings occurred at the site of application in male and female rats administered 175 or 350 mg/kg and included dermal irritation, thickened skin, and ulceration.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical findings occurred at the site of application in rats administered 175 or 350 mg/kg and included dermal irritation, thickened skin, and ulceration. Treatment-related lesions of the skin were limited to the site of application.

Mortality:

mortality observed, treatment-related

Description (incidence):

All core study rats survived to the end of the study. Treatment-related clinical findings occurred at the site of application in male and female rats administered 175 or 350 mg/kg and included dermal irritation, thickened skin, and ulceration.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The final mean body weights and mean body weight gains of 175 and 350 mg/kg males were significantly less than those of the vehicle controls; the final mean body weights and body weight gains of dosed females were similar to those of the vehicle controls

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no changes in the haematology or serum chemistry variables attributable to methyl trans-styryl ketone administered dermally to rats for 14 weeks

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no changes in the haematology or serum chemistry variables attributable to methyl trans-styryl ketone administered dermally to rats for 14 weeks

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute thymus weights of 87.5, 175, and 350 mg/kg males were significantly less than that of the vehicle controls, and the relative liver weight of 350 mg/kg females was significantly greater than that of the vehicle controls.

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were increased incidences of epidermal hyperplasia, hyperkeratosis, chronic active inflammation, epidermal necrosis, and sebaceous gland hypertrophy in dosed groups of males / females. In females, there were also epidermal degeneration & ulceration.

Details on results:

CLINICAL SIGNS AND MORTALITY
All core study rats survived to the end of the study. Treatment-related clinical findings occurred at the site of application in male and female rats administered 175 or 350 mg/kg and included dermal irritation, thickened skin, and ulceration. Treatment-related lesions of the skin were limited to the site of application.

BODY WEIGHT AND WEIGHT GAIN
The final mean body weights and mean body weight gains of 175 and 350 mg/kg males were significantly less than those of the vehicle controls; the final mean body weights and body weight gains of dosed females were similar to those of the vehicle controls.

HAEMATOLOGY AND CLINICAL CHEMISTRY
There were no changes in the haematology or serum chemistry variables attributable to methyl trans-styryl ketone administered dermally to rats for 14 weeks.

ORGAN WEIGHTS
Absolute thymus weights of 87.5, 175, and 350 mg/kg males were significantly less than that of the vehicle controls, and the relative liver weight of 350 mg/kg females was significantly greater than that of the vehicle controls.

GROSS PATHOLOGY
Similar to rats, skin lesions in mice occurred at the site of application and included hyperkeratosis, chronic active inflammation, epidermal hyperplasia, and sebaceous gland hypertrophy. Nasal lesions were limited to atrophy of the olfactory epithelium in males and females and were likely due to methyl trans-styryl ketone volatilized from the site of application.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related lesions of the skin were limited to the site of application. There were increased incidences of epidermal hyperplasia, hyperkeratosis, chronic active inflammation, epidermal necrosis, and sebaceous gland hypertrophy in dosed groups of males and females. In females, there were also epidermal degeneration and ulceration. In most cases, there were increases in the mean severities of these lesions at 87.5 mg/kg and greater.
Epidermal hyperplasia was characterized by thickening of the epidermis due to increased layers of epidermal cells in the stratum spinosum and stratum granulosum: one to two cell layers are considered normal, three to four layers define minimal hyperplasia, five to six layers define mild hyperplasia, seven to eight layers define moderate hyperplasia, and more than eight layers define marked hyperplasia. Hyperkeratosis was characterized by increased thickness of the stratum corneum, which was composed of multiple layers of eosinophilic lamellar keratin. Epidermal necrosis was characterized by loss of cellular and nuclear detail and epidermal pallor with retention of the normal architecture. In some cases, there was also karyorrhectic debris. There were frequent subepidermal or subcorneal clefts filled with fluid, cellular debris, and intact and degenerate neutrophils. A serocellular crust composed of similar material, but lying on the surface of the lesion, was often associated with the necrotic areas. In severe cases, the necrosis extended into the dermis where there was increased eosinophilia and hyalinization of the extracellular matrix of the dermis and scattered foci of hemorrhage. A rim of degenerate neutrophils frequently delineated the deep margin of the necrotic dermis. In some cases, the necrosis also affected the follicular epithelium. Ulceration was defined by the complete loss of the epidermis with an overlying serocellular crust. Epidermal degeneration was characterized by swelling and vacuolation of the basal keratinocytes and variably sized, intraepidermal cystic spaces filled with fluid and occasional sloughed epidermal cells or neutrophils.
In severe cases, the degenerative changes extended into the hair follicles. Degenerative changes were seen in many treated animals, but the diagnosis was made only when it was considered the primary lesion. Chronic inflammation was characterized by the infiltration of varying numbers of lymphocytes and macrophages, which were also occasionally found in the subcutis and epidermis. In severe cases, there were regions of dermal fibroproliferation with loss of hair follicles, which was considered a component of chronic inflammation.
In the nose, there were significantly increased incidences of goblet cell hyperplasia of the respiratory epithelium in 350 mg/kg males and 22, 175 and 350 mg/kg females. This lesion consisted of increased numbers of goblet cells in the respiratory epithelium.

OTHER FINDINGS
There were no significant differences in any of the reproductive organ weights or sperm parameters of male rats, or in the estrous cyclicity of female rats, at any dose when compared to the vehicle controls.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

87.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

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 and Body Weights of Rats in the 3-Month Dermal Study of Methyl trans-Styryl Ketone^a
Dose (mg/kg)	Survival^b	initial Body Weight (g)	Final Body Weight (g)	Change in Body Weight (g)	Final Weight Relative to Controls(%)
Male
0	10/10	100±2	309 ± 6	209 ± 5
22	10/10	100±3	311 ± 7	211 ± 6	100
44	10/10	100 ± 3	308 ± 8	208 ± 6	99
87.5	10/10	101 ± 3	300 ± 8	200 ± 5	97
175	10/10	99±3	284±6*	185 ± 4**	92
350	10/10	99±3	278±7**	179 ± 5**	90
Female
0	10/10	94 ± 1	180 ± 4	86 ± 3
22	10/10	93 ± 2	180 ± 4	87 ± 3	100
44	10/10	93 ± 2	183 ± 4	90 ± 3	102
87.5	10/10	94 ± 2	174 ± 2	80 ± 2	97
175	10/10	93 ± 2	175 ± 3	83 ± 3	98
350	10/10	94 ± 1	176 ± 2	82 ± 3	98
*  Significantly different (P<0.05) from the vehicle control group by William's test
** P<0.01
^a  Weights and weight changes are given as mean ± standard error.
^b  Number of animals surviving at 14 weeks/number initially in group

TABLE 3
Incidences of Selected Nonneoplastic Lesions in Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketone
	Vehicle Control	22 mg/kg	44 mg/kg	87.5 mg/kg	175 mg/kg	350 mg/kg
Male
Nose^a	10	10	10	10	10	10
Respiratory Epithelium,
Hyperplasia, Goblet Cell^b	2 (1.0)^c	6 (1.3)	1 (1.0)	6 (1.2)	4 (1.3)	9** (1.7)
Skin, Site of Application	10	10	9	10	10	10
Sebaceous Gland,
Hypertrophy	0	1 (1.0)	3 (1.0)	6** (1.2)	10** (1.8)	7** (1.9)
Epidermis, Hyperplasia	0	3 (1.0)	6** (1.0)	8** (1.5)	10** (2.2)	10** (2.3)
Hyperkeratosis	1 (1.0)	6* (1.2)	7** (1.4)	10** (1.7)	10** (1.9)	9** (2.1)
Inflammation, Chronic Active	0	0	0	0	3 (1.7)	4*(2.5)
Necrosis	0	0	0	0	3 (2.0)	3 (2.3)
Female
Nose	10	10	10	10	10	10
Respiratory Epithelium,
Hyperplasia, Goblet Cell	0	8** (1.0)	3 (1.0)	3 (1.0)	7** (1.4)	5*(1.2)
Skin, Site of Application	10	10	10	10	10	10
Sebaceous Gland, Hypertrophy                         0                     1   (1.0)          5*  (1.0)          7** (1.0)          4*  (1.5)          8** (1.6)
Epidermis, Degeneration	0	0	0	0	2 (3.0)	1 (1.0)
Epidermis, Hyperplasia	0	3 (1.0)	3 (1.0)	7** (1.6)	9** (2.2)	10** (2.2)
Hyperkeratosis	0	6** (1.0)	6** (1.0)	9** (1.9)	8** (1.6)	9** (2.0)
Inflammation, Chronic Active	0	0	0	1 (1.0)	6**(2.5)	4* (2.8)
Necrosis	0	0	0	0	4* (2.0)
Ulcer	0	0	0	0	1	1
*  Significantly different (P<0.05) from the vehicle 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 Dermal Study of Methyl trans-Styryl Ketone^a
	Vehicle Control	22 mg/kg	44 mg/kg	87.5 mg/kg	175 mg/kg	350 mg/kg
Male
Haematology
n Day 4	10	10	10	9	10	10
Day 24	10	10	10	10	10	10
Week14	10	10	10	10	10	10
Haematocrit(%)
Day 4	38.5 ± 0.5	37.5 ± 1.0	38.3±0.3	38.4 ± 0.5	38.7 ± 0.2	38.6 ± 0.4
Day 24	43.8±0.5	43.9 ± 0.7	43.9 ± 0.4	43.7 ± 0.5	43.0 ± 0.3	43.4 ± 0.5
Week14	44.8 ± 0.4	44.1 ± 0.5	44.8 ± 0.4	44.6 ± 0.4	44.6 ± 0.7	44.8 ± 0.5
Haemoglobin (g/dL)
Day 4	13.2 ± 0.2	12.9 ± 0.3	13.2 ± 0.1	13.2 ± 0.1	13.3 ± 0.1	13.2 ± 0.1
Day 24	15.4 ± 0.2	15.5±0.2	15.4 ± 0.1	15.3±0.1	15.0 ± 0.1	15.2 ± 0.2
Week 14	15.6±0.1	15.4 ± 0.1	15.7 ± 0.1	15.6 ± 0.1	15.5±0.1	15.7 ± 0.1
Erythrocytes (10^6/uL)
Day 4	6.93 ± 0.08	6.78 ± 0.16	6.90 ± 0.07	6.92 ± 0.09	7.04 ± 0.04	7.07 ± 0.07
Day 24	8.02 ± 0.10	8.02 ± 0.11	8.03 ± 0.08	8.00 ± 0.10	7.88 ± 0.05	7.89 ± 0.09
Week 13	8.93 ± 0.06	8.87 ± 0.08	8.99 ± 0.04	8.93 ± 0.07	8.87 ± 0.09	8.98 ± 0.08
Reticulocytes (10^6/uL)
Day 4	0.59 ± 0.03	0.58 ± 0.03	0.60 ± 0.02	0.58 ± 0.01	0.56 ± 0.02	0.63 ± 0.01
Day 24	0.40±0.04	0.41 ± 0.03	0.41 ± 0.02	0.32 ± 0.01	0.37 ± 0.03	0.38 ± 0.03
Week 14	0.24 ± 0.02	0.19 ± 0.02	0.21 ± 0.02	0.25 ± 0.01	0.22 ± 0.02	0.25 ± 0.01
Mean cell volume (fL)
Day 4	55.4 ± 0.2	55.3 ± 0.3	55.5 ± 0.2	55.2 ± 0.2	54.8 ± 0.3	54.6 ± 0.3
Day 24	54.6±0.2	54.7 ± 0.2	54.7 ± 0.2	54.6 ± 0.2	54.7 ± 0.2	55.0 ± 0.2
Week 14	50.1 ± 0.2	49.7 ± 0.3	49.7 ± 0.4	49.9 ± 0.4	50.3 ± 0.3	49.8 ± 0.1
Mean cell hemoglobin (pg)
Day 4	19.0 ± 0.1	19.1 ± 0.1	19.2 ± 0.1	19.1 ± 0.1	18.9 ± 0.1	18.7 ± 0.1*
Day 24	19.1 ± 0.1	19.2 ± 0.1	19.2 ± 0.1	19.2 ± 0.1	19.0 ± 0.0	19.2 ± 0.1
Week 14	17.4 ± 0.1	17.4 ± 0.0	17.5 ± 0.0	17.5 ± 0.1	17.5 ± 0.1	17.5 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4	34.2 ± 0.1	34.4 ± 0.1	34.6 ± 0.1	34.5 ± 0.1	34.4 ± 0.1	34.2 ± 0.1
Day 24	35.0±0.1	35.2 ± 0.1	35.0 ± 0.1	35.0 ± 0.1	34.8 ± 0.1	34.9 ± 0.1
Week 14	34.8±0.2	34.9 ± 0.2	35.1 ± 0.2	35.0 ± 0.2	34.8 ± 0.2	35.0 ± 0.2
Platelets (10^3/uL)
Day 4	727.3 ± 14.5	706.1 ± 19.1	685.8 ± 35.6	745.6 ± 18.4	743.8 ± 9.6	742.7 ± 19.2
Day 24	563.8 ± 20.0	546.3 ± 23.8	536.1 ± 27.9	552.3 ± 21.6	569.5 ± 11.3	518.0 ± 27.7
Week 14	601.9 ± 10.9	514.0±19.5**	541.2 ± 19.9	535.5 ± 14.4*	546.6 ± 21.6	563.5 ± 16.9
Leukocytes (10^3/uL)
Day 4	8.72 ± 0.42	8.29 ± 0.56	9.43 ± 0.72	9.67 ± 0.59	10.54 ± 0.77	9.69 ± 0.64
Day 24	12.17 ± 0.54	11.71 ± 0.46	12.48 ± 0.35	12.26 ± 0.41	12.26 ± 0.29	12.06 ± 0.54
Week 14	7.80±0.44	7.62 ± 0.38	8.46 ± 0.31	8.90 ± 0.37	8.28 ± 0.32	7.94 ± 0.44
Segmented neutrophils (10^3/uL)
Day 4	0.79 ± 0.12	0.89 ± 0.08	0.94 ± 0.14	1.02 ± 0.12	1.48 ± 0.18**	1.95 ± 0.16**
Day 24	1.09 ± 0.14	0.88 ± 0.08	1.30 ± 0.14	1.13±0.17	1.27 ± 0.11	1.24 ± 0.12
Week 14	1.02 ± 0.13	0.98 ± 0.12	1.13±0.10	1.17 ± 0.09	1.16 ± 0.09	1.23 ± 0.12
Lymphocytes (10^3/uL)
Day 4	7.40±0.33	6.88 ± 0.49	7.89 ± 0.58	8.04 ± 0.51	8.33 ± 0.57	7.19 ± 0.50
Day 24	10.42 ± 0.46	10.05 ± 0.40	10.40 ± 0.33	10.45 ± 0.30	10.10 ± 0.30	10.12 ± 0.43
Week 14	6.48 ± 0.40	6.25 ± 0.32	6.89 ± 0.26	7.17 ± 0.32	6.71 ± 0.35	6.35 ± 0.38
Monocytes (10^3/uL)
Day 4	0.50±0.06	0.48 ± 0.08	0.58 ± 0.05	0.56 ± 0.08	0.71 ± 0.09	0.50 ± 0.07
Day 24	0.63 ± 0.11	0.72 ± 0.10	0.77 ± 0.09	0.65 ± 0.10	0.83 ± 0.06	0.64 ± 0.11
Week 14	0.22 ± 0.02	0.36 ± 0.07	0.34 ± 0.06	0.38 ± 0.08	0.30 ± 0.04	0.31 ± 0.06
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	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	0.04 ± 0.02	0.40 ± 0.02	0.03 ± 0.02	0.05 ± 0.03	0.02 ± 0.01	0.04 ± 0.02
Day 24	0.04 ± 0.02	0.07 ± 0.02	0.01 ± 0.01	0.04 ± 0.02	0.06 ± 0.03	0.06 ± 0.03
Week 14	0.05 ± 0.02	0.02 ± 0.01	0.08 ± 0.03	0.12 ± 0.02	0.06 ± 0.03	0.04 ± 0.01
Clinical Chemistry
n	10	10	10	10	10	10
Ureanitrogen(mg/dL)
Day 4	14.4 ± 0.6	14.6 ± 0.3	13.6 ± 0.6	14.6 ± 0.5	13.9 ± 0.4	14.9 ± 0.5
Day 24	13.1 ± 0.4	12.8 ± 0.4	12.5 ± 0.4	11.7 ± 0.6	12.6 ± 0.5	12.1 ± 0.4
Week 14	16.4 ± 0.5	16.8 ± 0.6	16.7 ± 0.6	17.2 ± 1.1	17.5 ± 0.5	18.3 ± 0.4*
Creatinine (mg/dL)
Day 4	0.34 ± 0.02	0.30 ± 0.00	0.32 ± 0.01	0.31 ± 0.01	0.32 ± 0.01	0.36 ± 0.02
Day 24	0.30 ± 0.00	0.33 ± 0.02	0.32 ± 0.01	0.32 ± 0.01	0.32 ± 0.01	0.30 ± 0.01
Week 14	0.43 ± 0.02	0.39 ± 0.02	0.41 ± 0.02	0.41 ± 0.01	0.44 ± 0.02	0.43 ± 0.02
Total protein (g/dL)
Day 4	6.0 ± 0.1	6.1 ± 0.1	6.1 ± 0.1	6.0 ± 0.1	6.2 ± 0.1	6.2 ± 0.1
Day 24	6.6 ± 0.1	6.5 ± 0.1	6.5 ± 0.0	6.5 ± 0.1	6.5 ± 0.0	6.3 ± 0.1**
Week 14	7.6 ± 0.1	7.4 ± 0.1	7.5 ± 0.1	7.5 ± 0.1	7.4 ± 0.1	7.4 ± 0.1
Albumin (g/dL)
Day 4	4.1 ± 0.1	4.1 ± 0.1	4.1 ± 0.1	4.1 ± 0.1	4.2 ± 0.1	4.2 ± 0.0
Day 24	4.3 ± 0.0	4.3 ± 0.0	4.3 ± 0.0	4.3 ± 0.0	4.3 ± 0.0	4.2 ± 0.0*
Week 14	4.8 ± 0.0	4.7 ± 0.1	4.8 ± 0.1	4.8 ± 0.1	4.8 ± 0.1	4.7 ± 0.1
Globulin (g/dL)
Day 4	1.9 ± 0.0	2.0 ± 0.1	2.0 ± 0.0	1.9 ± 0.0	2.0 ± 0.1**	2.0 ± 0.0**
Day 24	2.3 ± 0.0	2.2 ± 0.1	2.2 ± 0.0	2.2 ± 0.0	2.2 ± 0.0	2.1 ± 0.0*
Week 14	2.9 ± 0.1	2.7 ± 0.1	2.8 ± 0.1	2.7 ± 0.1	2.6 ± 0.1	2.7 ± 0.1
Albumin/globulin ratio
Day 4	2.2 ± 0.0	2.1 ± 0.1*	2.1 ± 0.0**	2.2 ± 0.0*	2.1 ± 0.1**	2.1 ± 0.0**
Day 24	1.9 ± 0.0	2.0 ± 0.0	1.9 ± 0.0	2.0 ± 0.0	2.0 ± 0.0	2.0 ± 0.0
Week 14	1.7 ± 0.0	1.8 ± 0.0	1.7 ± 0.0	1.7 ± 0.0	1.8 ± 0.0	1.8 ± 0.0
Alanine aminotransferase(IU/L)
Day 4	55 ± 1	58 ± 1	59 ± 2	59 ± 2	60 ± 2	60 ± 2
Day 24	48 ± 1	46 ± 1	45 ± 1	47 ± 1	47 ± 1	48 ± 1
Week 14	67 ± 5	62 ± 3	61 ± 2	64 ± 4	63 ± 4	58 ± 2
Alkaline phosphatase (IU/L)
Day 4	698 ± 13	701 ± 17	698 ± 21	705 ± 13	685 ± 15	645 ± 10*
Day 24	511 ± 9	504 ± 14	501 ± 12	488 ± 7	473 ± 9*	468 ± 12**
Week 14	228 ± 3	229 ± 4	227 ± 4	231 ± 5	236 ± 5	228 ± 5
Creatine kinase (IU/L)
Day 4	413 ± 25	584 ± 73	751 ± 119*	715 ± 110*	507 ± 80	638 ± 112
Day 24	307 ± 42	336 ± 69	367 ± 49	312 ± 28	346 ± 43	318 ± 26
Week 14	171 ± 15	192 ± 23	212 ± 30	183 ± 14	268 ± 81	196 ± 25
Sorbitol dehydrogenase (IU/L)
Day 4	23 ± 1	23 ± 1	21 ± 2	22 ± 1	20 ± 2	21 ± 2
Day 24	22 ± 1	19±1	20 ± 1	21 ± 1	19 ± 1	22 ± 1
Week 14	22 ± 1	21 ± 1	22 ± 1	24 ± 2	22 ± 2	23 ± 1
Bile salts (umol/L)
Day 4	26.7 ± 1.4	28.5 ± 2.4	31.4 ± 2.1	26.5±1.7	29.9 ± 1.2	30.3±3.0
Day 24	26.4±2.4	24.6 ± 1.9	24.6 ± 2.6	24.5±1.8	27.7 ± 2.4	25.9 ± 1.7
Week 14	35.1 ± 4.0	26.9 ± 2.9	22.9 ± 3.0	29.6 ± 3.9	31.8±2.7	27.4 ± 2.6
Female
n
Day 4	10	9	10	10	10	10
Day 24	10	10	10	9	10	10
Week 14	10	10	10	10	10	10
Hematology
Hematocrit (%)
Day 4	42.8±0.7	44.4 ± 1.2	43.1 ± 0.7	44.3 ± 1.4	43.4 ± 1.1	42.8±1.0
Day 24	44.9 ± 0.3	45.1 ± 0.4	47.0 ± 0.7*	46.0 ± 0.4	45.6 ± 0.2	45.3±0.4
Week 14	39.0±0.8	38.7 ± 0.5	38.5 ± 0.6	39.2 ± 0.5	39.3±0.4	38.5±0.7
Hemoglobin (g/dL)
Day 4	14.9 ± 0.2	15.4 ± 0.4	14.9 ± 0.2	15.4 ± 0.5	15.0 ± 0.4	14.9 ± 0.3
Day 24	15.9 ± 0.1	16.0 ± 0.1	16.6 ± 0.2*	16.3±0.2	16.0 ± 0.1	16.1 ± 0.2
Week 14	15.1 ± 0.2	15.0 ± 0.2	15.1 ± 0.1	15.4 ± 0.2	15.5±0.2	15.0±0.3
Erythrocytes (10^6/uL)
Day 4	7.74 ± 0.11	8.02 ± 0.23	7.79 ± 0.10	8.03 ± 0.29	7.82 ± 0.17	7.73±0.17
Day 24	8.28 ± 0.06	8.23±0.08	8.57 ± 0.12	8.40 ± 0.07	8.35 ± 0.05	8.32 ± 0.07
Week 14	7.89 ± 0.12	7.82 ± 0.10	7.88 ± 0.10	7.99 ± 0.12	8.07 ± 0.08	7.87 ± 0.12
Reticulocytes (10^6/uL)
Day 4	0.59 ± 0.02	0.55 ± 0.04	0.53 ± 0.02	0.58 ± 0.05	0.54 ± 0.03	0.54 ± 0.03
Day 24	0.25 ± 0.01	0.27 ± 0.02	0.22 ± 0.02	0.23 ± 0.02	0.24 ± 0.02	0.22 ± 0.02
Week 14	0.22 ± 0.02	0.21 ± 0.02	0.23 ± 0.01	0.21 ± 0.01	0.21 ± 0.02	0.20 ± 0.02
Mean cell volume (fL)
Day 4	55.2 ± 0.1	55.3 ± 0.2	55.4 ± 0.3	55.6 ± 0.2	55.5±0.2	55.4 ± 0.2
Day 24	54.4 ± 0.2	54.7 ± 0.2	54.9 ± 0.2	54.8 ± 0.2	54.6 ± 0.3	54.4 ± 0.2
Week 14	49.3±0.4	49.5 ± 0.4	48.8±0.3	48.9 ± 0.5	48.7 ± 0.3	49.1 ± 0.4
Mean cell hemoglobin (pg)
Day 4	19.2 ± 0.1	19.2 ± 0.1	19.2 ± 0.1	19.3 ± 0.1	19.2 ± 0.1	19.3 ± 0.1
Day 24	19.2 ± 0.1	19.4 ± 0.1	19.4 ± 0.1	19.4 ± 0.1	19.2 ± 0.1	19.3 ± 0.1
Week 14	19.2 ± 0.1	19.2 ± 0.1	19.2 ± 0.1	19.2 ± 0.1	19.2 ± 0.1	19.1 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4	34.8±0.1	34.7 ± 0.1	34.6 ± 0.1	34.8 ± 0.1	34.6 ± 0.0	34.8±0.0
Day 24	35.5 ± 0.1	35.4 ± 0.1	35.3±0.1	35.4 ± 0.1	35.2 ± 0.1*	35.4 ± 0.1
Week 14	38.9 ± 0.4	38.7 ± 0.4	39.3±0.3	39.2 ± 0.4	39.3±0.3	39.0 ± 0.3
Platelets (10^3/uL)
Day 4	688.0±30.2	637.1 ± 27.6	663.8 ± 23.5	694.5 ± 13.9	689.4 ± 16.8	666.9 ± 22.8
Day 24	609.6 ± 15.7	627.2 ± 18.9	543.9 ± 41.9	575.3 ± 24.4	548.1 ± 16.1	552.8±26.3
Week 14	567.4 ± 20.7	593.8 ± 23.7	614.9 ± 19.3	611.8±24.1	593.2 ± 14.8	562.5 ± 30.4
Leukocytes(10^3/uL)
Day4	11.99 ± 0.59	11.13±0.59	10.29 ± 0.50	10.27 ± 0.40	11.93 ± 0.77	12.02 ± 0.45
Day24	12.59 ± 0.46	11.59 ± 0.39	11.36 ± 0.64	12.20 ± 0.62	11.87 ± 0.68	12.88 ± 0.49
Week 14	5.02 ± 0.55	4.94 ± 0.37	5.83 ± 0.29	5.91 ± 0.38	6.16 ± 0.39	5.61 ± 0.44
Segmented neutrophils (10^3/uL)
Day 4	1.17 ± 0.13	0.94 ± 0.07	0.81 ± 0.09	0.72 ± 0.08*	1.21 ± 0.12	1.27 ± 0.14
Day 24	1.01 ± 0.09	1.22 ± 0.11	0.96 ± 0.08	1.14 ± 0.16	1.10 ± 0.10	1.44 ± 0.11*
Week 14	0.50±0.07	0.56±0.03	0.63 ± 0.09	0.85 ± 0.15*	0.81 ± 0.10*	0.82 ± 0.09**
Lymphocytes (10^3/uL)
Day 4	10.16±0.52	9.59 ± 0.53	8.82 ± 0.45	8.99 ± 0.32	10.05±0.73	10.08 ± 0.42
Day 24	10.60±0.37	9.56±0.36	9.75 ± 0.63	10.33±0.60	10.06 ± 0.63	10.80 ± 0.49
Week 14	4.22 ± 0.45	4.16±0.34	4.96 ± 0.21	4.78 ± 0.31	5.11 ± 0.31	4.53 ± 0.41
Monocytes (10^3/uL)
Day 4	0.66±0.10	0.59 ± 0.08	0.64 ± 0.10	0.53 ± 0.09	0.63 ± 0.06	0.66 ± 0.09
Day 24	0.88 ± 0.12	0.74 ± 0.08	0.59 ± 0.08	0.67 ± 0.08	0.66 ± 0.08	0.57 ± 0.08
Week 14	0.26±0.04	0.19 ± 0.04	0.19 ± 0.03	0.21 ± 0.04	0.18 ± 0.04	0.23 ± 0.04
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	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	0.01 ± 0.01	0.01 ± 0.01	0.02 ± 0.01	0.02 ± 0.01	0.04 ± 0.02	0.01 ± 0.01
Day 24	0.11 ± 0.04	0.07 ± 0.02	0.06 ± 0.02	0.06 ± 0.03	0.06 ± 0.02	0.07 ± 0.03
Week 14	0.03 ± 0.02	0.02 ± 0.01	0.04 ± 0.02	0.04 ± 0.02	0.06 ± 0.02	0.02 ± 0.01
Clinical Chemistry
Urea nitrogen (mg/dL)
Day 4	13.6±0.7	14.6±1.0	14.3 ± 0.7	14.3±0.6	13.0 ± 0.7	13.4 ± 0.4
Day 24	15.7 ± 0.3	15.6 ± 0.7	15.6 ± 0.7	16.3±0.6	15.8 ± 0.4	15.2 ± 0.4
Week 14	18.8±0.8	19.1 ± 0.7	18.4 ± 0.6	17.6 ± 0.7	19.0 ± 0.6	18.9 ± 1.1
Creatinine (mg/dL)
Day 4	0.34 ± 0.02	0.32 ± 0.01	0.30 ± 0.01	0.34 ± 0.02	0.31 ± 0.02	0.36 ± 0.02
Day 24	0.30±0.00	0.32 ± 0.01	0.35 ± 0.02	0.30 ± 0.00	0.35 ± 0.02	0.31 ± 0.02
Week 14	0.42 ± 0.01	0.45 ± 0.02	0.42 ± 0.01	0.42 ± 0.01	0.44 ± 0.02	0.42 ± 0.02
Total protein (g/dL)
Day 4	6.3 ± 0.1	6.2 ± 0.1	6.3 ± 0.1	6.4 ± 0.1	6.2 ± 0.1	6.3 ± 0.1
Day 24	6.4 ± 0.1	6.4 ± 0.1	6.6 ± 0.1	6.6 ± 0.1	6.7 ± 0.1*	6.4 ± 0.1
Week 14	6.8 ± 0.1	7.1 ± 0.1	7.0 ± 0.1	7.0 ± 0.1	7.1 ± 0.1	6.8 ± 0.1
Albumin (g/dL)
Day 4	4.4 ± 0.0	4.4 ± 0.1	4.5 ± 0.0	4.4 ± 0.1	4.4 ± 0.1	4.4 ± 0.0
Day 24	4.4 ± 0.0	4.5 ± 0.0	4.5 ± 0.1	4.6 ± 0.0	4.6 ± 0.0**	4.5 ± 0.1
Week 14	4.6±0.0	4.8 ± 0.1	4.8 ± 0.1	4.7 ± 0.1	4.7 ± 0.1	4.6 ± 0.1
Globulin (g/dL)
Day 4	1.9 ± 0.1	1.8±0.0	1.9 ± 0.1	2.0 ± 0.1	1.9 ± 0.1	1.9 ± 0.1
Day 24	2.0±0.0	2.0 ± 0.0	2.0 ± 0.0	2.1 ± 0.0	2.0 ± 0.0	2.0 ± 0.0
Week 14	2.2 ± 0.0	2.3 ± 0.0	2.2 ± 0.1	2.3 ± 0.1	2.3 ± 0.1	2.2 ± 0.1
Albumin/globulin ratio
Day 4	2.3 ± 0.1	2.4 ± 0.0	2.4 ± 0.1	2.3 ± 0.1	2.3±0.1	2.4±0.0
Day 24	2.3 ± 0.1	2.3±0.0	2.2 ± 0.0	2.2±0.0	2.3±0.0	2.3±0.1
Week 14	2.1 ± 0.0	2.1 ± 0.0	2.2 ± 0.1	2.1 ± 0.1	2.0±0.0	2.2 ± 0.1
Alanine aminotransferase (IU/L)
Day 4	47 ± 2	48 ± 1	46 ± 1	54 ± 3	47 ± 1	53 ± 2
Day 24	40 ± 1	38 ± 2	41 ± 2	37 ± 1	39 ± 1	40 ± 1
Week 14	61 ± 5	81 ± 10	64 ± 4	57 ± 4	62 ± 3	59 ± 2
Alkaline phosphatase (IU/L)
Day 4	581 ± 17	565 ± 13	561 ± 8	567 ± 8	551 ± 17	551 ± 10
Day 24	390 ± 4	393 ± 10	400 ± 9	389 ± 11	391 ± 7	403 ± 12
Week 14	194 ± 3	188 ± 4	186±5	195 ± 6	186±5	184 ± 5
Creatine kinase (IU/L)
Day 4	736 ± 257	570±139	493 ± 94	1,281 ± 404	452 ± 73	602 ± 84
Day 24	295 ± 30	378 ± 103	231 ± 24	350 ± 56	253±25	448 ± 112
Week 14	251 ± 48	198 ± 25	212 ± 24	226 ± 29	225 ± 37	210 ± 38
Sorbitol dehydrogenase (IU/L)
Day 4	15±2	18 ± 2	15±2	13±2	17 ± 2	13 ± 2
Day 24	22 ± 1	21 ± 1	23 ± 2	20 ± 1	22 ± 1	21 ± 2
Week 14	22 ± 2	28 ± 2*	25 ± 1	24 ± 2	25 ± 2	21 ± 1
Bile salts(umol/L)
Day 4	25.3±2.0	27.7 ± 4.3	23.8±1.1	25.7 ± 2.6	22.5±1.9	23.9 ± 3.1
Day 24	26.4 ± 2.2	23.2 ± 2.3	20.3±1.8	19.9 ± 1.8	20.8 ± 2.1	24.9 ± 2.5
Week 14	34.9 ± 3.2	36.9 ± 2.9	38.5 ± 5.4	32.5 ± 2.6	31.3 ± 3.4	35.8±2.7
*  Significantly different (P<0.05) from the vehicle 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.

TABLE 5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketone^a
	Vehicle Control	22mg/kg	44mg/kg	87.5mg/kg	175mg/kg	350mg/kg
Male
n	10	10	10	10	10	10
Necropsy body wt	309±6	311±7	308 ± 8	300 ± 8	284 ± 6*	278 ± 7**
Heart
Absolute	0.92±0.02	0.94±0.03	0.92 ± 0.02	0.90 ± 0.03	0.88 ± 0.01	0.86 ± 0.02
Relative	2.985±0.041	3.034±0.051	2.990 ± 0.035	2.980 ± 0.044	3.087 ± 0.049	3.100±0.049
R. Kidney
Absolute	1.03±0.03	1.02±0.02	1.01±0.03	1.00±0.02	0.95 ± 0.02	0.96 ± 0.02
Relative	3.312±0.040	3.282 ± 0.036	3.279 ± 0.033	3.331 ± 0.043	3.362 ± 0.065	3.454 ± 0.049
Liver
Absolute	10.92±0.30	11.53±0.38	11.26±0.50	11.02±0.39	10.25 ± 0.25	9.90 ± 0.22
Relative	35.274±0.495	37.074 ± 0.503	36.439±0.748	36.640 ± 0.577	36.102±0.396	35.687 ± 0.321
Lung
Absolute	1.42 ± 0.05	1.46 ± 0.05	1.41 ± 0.07	1.30±0.04	1.31±0.06	1.37±0.07
Relative	4.582 ± 0.150	4.708 ± 0.190	4.573 ± 0.208	4.315±0.066	4.631 ± 0.177	4.943 ± 0.253
R. Testis
Absolute	1.410 ± 0.025	1.397 ± 0.029	1.363 ± 0.030	1.377 ± 0.019	1.333±0.026	1.342 ± 0.025
Relative	4.561 ± 0.044	4.504 ± 0.058	4.438 ± 0.067	4.602 ± 0.085	4.714±0.125	4.848 ± 0.083*
Thymus
Absolute	0.269 ± 0.011	0.254 ± 0.012	0.273 ± 0.009	0.238±0.010*	0.229 ± 0.005**	0.223 ± 0.011**
Relative	0.867 ± 0.028	0.816±0.022	0.890 ± 0.028	0.798 ± 0.044	0.807 ± 0.020	0.799 ± 0.029
Female
n	10	9	10	10	10	10
Necropsy body wt	180±4	180± 4^b	183±4	174 ± 2	175 ± 3	176 ± 2
Heart
Absolute	0.66±0.01	0.66 ± 0.02	0.67 ± 0.01	0.67 ± 0.01	0.65 ± 0.01	0.67 ± 0.01
Relative	3.689 ± 0.063	3.681 ± 0.065	3.644 ± 0.054	3.854 ± 0.068	3.729 ± 0.063	3.778 ± 0.033
R. Kidney
Absolute	0.73±0.03	0.70 ± 0.02	0.75 ± 0.02	0.75 ± 0.02	0.72 ± 0.02	0.75 ± 0.02
Relative	4.043±0.133	3.879 ± 0.052	4.074 ± 0.078	4.309 ± 0.131	4.091 ± 0.093	4.292 ± 0.116
Liver
Absolute	6.11±0.20	6.25 ± 0.18	6.37 ± 0.16	6.11 ± 0.11	6.16±0.15	6.35 ± 0.10
Relative	33.963 ± 0.728	34.688 ± 0.352	34.705±0.442	35.220 ± 0.437	35.102±0.525	36.106±0.393**
Lung
Absolute	1.10±0.04	1.08 ± 0.05	1.09±0.03	1.06±0.05	1.06±0.03	1.06±0.03
Relative	6.107±0.213	5.991 ± 0.233	5.961 ± 0.111	6.114±0.241	6.062 ± 0.171	6.030 ± 0.138
Thymus
Absolute	0.225 ± 0.012	0.226 ± 0.010	0.224 ± 0.007	0.206 ± 0.006	0.225 ± 0.011	0.215±0.007
Relative	1.253 ± 0.069	1.255 ± 0.033	1.221 ± 0.040	1.187±0.036	1.285 ± 0.063	1.223 ± 0.032
*  Significantly different (P<0.05) from the vehicle control group by Williams' test
** P<0.01
^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=10

Table 6 Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketone^a
	Vehicle Control	87.5 mg/kg	175 mg/kg	350 mg/kg
n	10	10	10	10
Weights (g)
Necropsy body wt	309 ± 6	300 ± 8	284±6**	278 ± 7**
L. Cauda epididymis	0.1669±0.0105	0.1537±0.0037	0.1471 ± 0.0042	0.1446±0.0064
L. Epididymis	0.4256 ± 0.0054	0.4358 ± 0.0076	0.4262 ± 0.0100	0.4017±0.0076
L. Testis	1.4617±0.0287	1.4516±0.0237	1.4483 ± 0.0242	1.3924 ± 0.0290
Spermatid measurements
Spermatid heads (10^6/testis)	171.38 ± 7.95	169.00 ± 6.25	174.25 ± 8.45	155.38±8.39
Spermatid heads (10^3/mg testis)	123.0 ± 4.5	123.3 ± 4.7	126.9 ± 4.7	118.4±5.5
Epididymalspermatozoal measurements
Sperm motility (%)	84.1 ± 0.8	85.3 ± 1.4	83.9 ± 0.8	83.8 ± 0.7
Sperm (10^6/cauda epididymis)	55.4 ± 10.4	46.3 ± 9.4	47.4 ± 8.3	36.6 ± 5.5
Sperm (10^3/mg cauda epididymis)	333 ± 62	301 ± 63	333 ± 63	255 ± 38
** Significantly different (P<0.01) from the vehicle control group by Williams' test
^a  Data are presented as mean ± standard error. Differences from the vehicle 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 Dermal Study of Methyl trans-Styryl Ketone^a
	Vehicle Control	87.5 mg/kg	175 mg/kg	350 mg/kg
Number weighed at necropsy	10	10	10	10
Necropsy body wt (g)	180 ± 4	174 ± 2	175 ± 3	176 ± 2
Proportion of regular cycling females^b	10/10	9/10	10/10	10/10
Estrous cycle length (days)	5.0 ± 0.05	4.9±0.08	5.0 ± 0.00	4.9 ± 0.07
Estrous stages (% of cycle)
Diestrus	59.2	57.5	60.0	62.5
Proestrus	17.5	15.0	14.2	15.8
Estrus	20.8	25.8	25.8	20.0
Metestrus	2.5	1.7	0.0	1.7
^a  Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnett's test (body weight) or Dunn's test (estrous cycle length). By multivariate analysis of variance, dosed females do not differ significantly from the vehicle 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 vehicle control group and each dosed group indicated there were no significant differences.
^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 TG411 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 dermal irritation, thickened skin and ulceration at the site of application. A NOAEL of 87.5 mg/kg bwwas derived.

Executive summary:

The substance methyl trans-styryl ketone was investigated for its repeated dose toxicity via the dermal route in rats (NTP, 2011). Groups of 10 male and 10 female rats were dermally administered 0, 22, 44, 87.5, 175, or 350 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 14 weeks. Groups of 10 male and 10 female clinical pathology rats were administered the same doses for 23 days. All rats survived to the end of the study. Mean body weights of 175 and 350 mg/kg males were significantly less than that of the vehicle controls. Clinical findings in groups administered 175 or 350 mg/kg included dermal irritation, thickened skin, and ulceration at the site of application. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male or female rats at the doses used in this study. Histologically, there were significantly increased incidences of epidermal hyperplasia, hyperkeratosis, chronic active inflammation, epidermal necrosis, and sebaceous gland hypertrophy in the skin at the site of application in males and/or females. There were significantly increased incidences of goblet cell hyperplasia of the nose in 350 mg/kg males and 22, 175, and 350 mg/kg females. So a NOAEL of 87.5 mg/kg bw can be derived.