Registration Dossier

Diss Factsheets

Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.

REACH

4-phenylbutenone

EC number: 204-555-1 | CAS number: 122-57-6

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

1) Japan MHLW, 2011, rats, oral, 28 days, NOAEL 60 mg/kg bw for males and females

2) NTP, 2012a, rats, oral, 3 month, NOAEL 145 mg/kg bw for females and 150 mg/kg bw for males
3) NTP, 2012a, mice, oral, 3 month, NOAEL 350 mg/kg bw for females and 400 mg/kg bw for males
4) NTP, 2012a, rats, dermal, 3 month, NOAEL 87.5 mg/kg bw for males and females
5) NTP, 2012a, mice, dermal, 3 month, LOAEL 87.5 mg/kg bw for males and females

Key value for chemical safety assessment

Toxic effect type:: dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen all close all

Reference 1

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.
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
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
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
Dose / conc.:: 0.025 other: %
Remarks:: approx. 18 and 19 mg/kg bw in females and males, respectively
Basis:
nominal in diet
Dose / conc.:: 0.05 other: %
Remarks:: approx. 36 and 38mg/kg bw in females and males, respectively
Basis:
nominal in diet
Dose / conc.:: 0.1 other: %
Remarks:: approx. 72 and 77 mg/kg bw in females and males, respectively
Basis:
nominal in diet
Dose / conc.:: 0.2 other: %
Remarks:: approx. 145 and 150 mg/kg bw in females and males, respectively
Basis:
nominal in diet
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
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.
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).
: 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
: 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
Critical effects observed:: not specified
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).

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-02-03 - 2011-06-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

not specified

Remarks:

the specific pages are missing, but it is assumed that the test was performed according to GLP criteria.

Limit test:

Species:

rat

Strain:

other: Sprague-Dawley SPF rats (Crl: CD (SD))

Details on species / strain selection:

Toxicity study guidelines require testing on rats. The strain of rats used for this study was chosen as their nature is well-known and rich background data are available.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 6 weeks
- Weight at study initiation: 191 to 226 g for males and 158 to 190 g for females
- Fasting period before study: not specified
- Housing: animal room
- Diet (e.g. ad libitum): ad libitum,solid feed CRF-1 via stainless steel feeder
- Water (e.g. ad libitum): ad libitum, City tap water via water bottle
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 25°C (setup range: 23 ± 3°C)
- Humidity (%): 43 to 58% (setup range: 50±20%)
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12-hour lighting per day (07:00 to 19:00)

Route of administration:

oral: gavage

Details on route of administration:

Administration by oral gavage using stomach sonde (between 08:05 and 11:41).

Vehicle:

methylcellulose

Remarks:

0.5 w/v% methyl cellulose aqueous solution

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
A required amount of the test substance was weighed accurately for each concentration and suspended in the 0.5 w/v% methyl cellulose aqueous solution to obtain 2.4 mg/mL solution (low dose group solution), 12 mg/mL solution (middle dose group solution) and 60 mg/mL solution (high dose group liquid). The test solution was prepared at least once a week and used within 7 days after preparation.
The test formulations were divided into one-day's aliquots, dispensed into brown glass bottles and stored in a cold place (in a refrigerator, measured temperature: 3 to 6°C) until use.

The dose volume was set at 10 mL/kg body weight. Individual dose volume was calculated based on the animal's most recently body weight.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): suitable vehicle
- Concentration in vehicle: 0.5 w/v% methyl cellulose aqueous solution
- Amount of vehicle (if gavage): 2.4 mg/mL solution (low dose group solution), 12 mg/mL solution (middle dose group solution) and 60 mg/mL solution (high dose group liquid.

Analytical verification of doses or concentrations:

yes

Remarks:

HPLC analysis method

Details on analytical verification of doses or concentrations:

For each concentration of the test solution used for administration at 1 week and 4 weeks, its concentration and homogeneity was confirmed by using HPLC analysis method. As a result, the ratio of the concentration to the nominal value was 94.6 to 107.7% (tolerance: 100±10%) and coefficient of variations (CV) ranged from 0.0 to 3.3% (tolerance: CV<10%), both of which met acceptance criteria.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once a day (7 times per week), followed by a 14 day recovery period with no administration

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

6 males and 6 females in group 12 mg/kg and 60 mg/kg
12 males and 12 females in control group and 300 mg/kg

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
According to the results and main changes of the preliminary 14-day oral toxicity study in rats (dose levels: 0 (0.5 w/v% MC solution), 100, 300 and 1000 mg/kg/day, 5 males and 5 females/group), changes in body weight and food intake as well as hematology examination, organ weight and necropsy in 1000 mg/kg (both male and female), toxicity in necropsy was observed in 300 mg/kg administration group for both sexes; In the 100 mg/kg administration group, changes in autopsy were observed, but only in males.
Therefore, in this study, the high dose level was set at 300 mg/kg, the middle and low dose level are 60 mg/kg and 12 mg/kg.

Positive control:

Observations and examinations performed and frequency:

Observations and examinations were conducted as indicated below.
Day 1 of administration: Starting day of administration
Week 1 of administration: From Day 1 to Day 7 of administration
Day 1 of recovery: Starting day of recovery (the day following the final administration)
Week 1 of recovery: From Day 1 to Day 7 of recovery

Clinical Observation
All animals were observed during administration 3 times a day, before administration, right after administration and around 2 hours after administration (however, detailed clinical observation of general condition, manipulative test, grip strength and motor activity measurement are carried out before administration and immediately after administration), and once a day during the recovery period for general observation including external appearance, nutritional condition, posture, behavior and excrement.

Detailed Clinical Observation, Manipulative Test, and Measurements of Grip Strength and Motor Activity
Detailed clinical observation was done once before the start of administration, and once a week during the administration and during the recovery period for all animals. The manipulative test and measurements of grip strength and motor activity were conducted on all animals in Week 4 (on Day 26 for males and Day 27 for females) of administration and in week 2 of recovery (day 12).
For the detailed clinical observation and manipulative test, measured values or scores were used for the evaluation. In addition, observation and examination were performed with the administration information restricted (blinded method) and animals arranged at random.

Detailed Clinical Observation
1) Home cage observation
Posture, convulsion, abnormal behavior
2) In-the-hand observations
Easiness of removal from cage, condition of fur and skin, secretions from eyes and nose,
Easiness, visible mucous membrane, autonomic nervous function (lacrimation, salivation, piloerection, pupil size, nirvana, and abnormal respiration), response to handling.
3) Open field observation
Arousal state, convulsion, abnormal behavior, stereotypy behavior, gait, posture, grooming, rising times, excretion (defecation count, urination)

Manipulative Test
Auditory response, approach response, touch response, Pain-sensitive reaction, pupillary reflex, aerial righting reflex, foot opening width when landing.

Grip Strength
Grip strength of forelimbs and hind limbs was measured by CPU gauge MODEL-9502A.

Motor Activity
Motor activity was measured by a motor activity sensor for experimental animals NS-AS01. Measurement was done for 1 hour, and measured values in 10-minute intervals and from 0 to 60 minutes were collected.

Measurement of Body Weight
All animals were weighed before administration on Day 1, 4, 7, 10, 14, 17, 21, 24 and 28 of administration and on Day 1, 3, 7, 10 and 14 of recovery. Body weight was measured between 08:40 and 9:50. On the day of necropsy, animals were weighed after fasting for approximately 16 hours from the previous day in order to calculate the relative organ weight (between 07:59 and 08:19).

Urinalysis (incl. water intake)
Urinalysis was conducted in Week 4 (23rd day) of administration and week 2 (9 day) of recovery.
For all animals in the main and recovery groups, animals were individually placed in cages each equipped with a urine collector. Then, 4-hour urine samples were collected from animals under fasting but with free access to water, followed by collection of 20-hour urine samples from animals with free access to food and water.
Items: appearance, glucose, pH, occult blood, ketones, protein, bilirubin, urobilinogen, sediment and urine volume (4 hour amount). For 20-h the test items are urine volume (20 hour amount) and Infiltration.

Hematological Examination
At the time of scheduled necropsy on the day after the end of the administration and recovery period, all animals which were fasted overnight (for approximately 16 to 21 hours) from the previous day were subjected to laparotomy under ether anesthesia, and blood (approximately 1 mL) was collected via the abdominal aorta into blood collecting tubes (SB-41) containing EDTA-2K for examination (Item I). In addition, blood samples (approximately 0.9 mL) were collected into tubes containing 3.8% sodium citrate solution (one volume to nine volumes of blood), and plasma samples obtained by centrifugation (3,100 rpm, 1,690 x g, 12 minutes) were examined for items II.
Items I: red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte count (Retic), platelet count (PLT), white blood cell count (WBC), white blood cell percentage.

Items II: prothrombin time (PT), activated partial thromboplastin time (APTT), Fibrinogen content (FIB).

Blood Chemistry Examination
Blood samples (approximately 4 mL) were collected at the same time as for those for hematological examination.

Test items for serum: ALP, total cholesterol (T-CHO), triglyceride (TG), Phospholipids (PL), total bilirubin (T-BIL), glucose (GLU), blood urea nitrogen (BUN), Na, K, Cl, Ca, P, total protein (TP), albumin (ALB), AJG ratio (A/G).
Test items for plasma: AST, ALT, LDH, γ-GTP.

Sacrifice and pathology:

Pathological Examinations
Necropsy
All animals scheduled for necropsy were sacrificed by exsanguinations via the abdominal aorta after blood collection. Macroscopic examination (by visual) was then performed on the organs/tissues throughout the body, including the external appearance and those in the cephalic, thoracic and abdominal cavities, and the results recorded.

Measurement of Organ Weight
For all animals, the weighed (absolute weight) of the following organs was measured, and the relative weight per 100 g body weight was calculated from the absolute weight and the body weight at the time of examination. For paired organs (marked with *), the right and left organs were weighed separately, but evaluation was done on the total of both sides.
Brain, adrenal gland *, thymus, tongue, heart, liver, kidney gradient, testis *, epididymis *, ovary *, uterus

Histopathological Examination
The organs/tissues below were collected and examined.

cerebrum, cerebellum, spinal cord (thoracic), sciatic nerve*, eyeball*, pituitary, thyroid*, Parathyroid gland *, adrenal*, thymus, spleen, mandibular lymph node, mesenteric lymph node, heart, trachea, lung (including bronchus), stomach, duodenum, jejunum, ileum (including Peyer's patches), cecum, colon, rectum, liver, kidney*, urinary bladder, testis *, Epididymis *, prostate, ovary*, uterus, sternum (including bone marrow), femur (including bone marrow) *, femoral skeletal muscle * and macroscopic lesions places.
In addition, the optic nerve *, Harder's gland *, thoracic aorta, tongue, esophagus, submandibular gland *, sublingual gland *, pancreas, vagina , Seminal vesicle, mammary gland (inguinal part) *, skin (inguinal part) *, individually identified part (auricle) and larynx were excised and preserved.

Statistics:

Statistical Analysis
Quantitative items for observation in open field, quantitative items in manipulative test,, grip strength measurement, measurement of motor activity, body weight (including weight gain), food consumption, water consumption, urinalysis, hematology test, blood chemistry test and organ weight data were statistically analyzed between the control group and each administration group.
For the main group, first, analysis of variance was conducted by the Bartlett test (level of significance: 1%). If variances were homogeneous, data were analyzed by the Dunnett method, whereas heterogeneous data were analyzed by meanrank test of Dunnett type between the control group and each dose group (levels of significance: 5 and 1%, two-tailed).
For the recovery groups, homogeneity of variance was tested for each group by the F -test (level of significance: 5%). For homogeneous data, the difference in the mean values between the control and treatment group was analyzed by the Student's t-test (levels of significance: 5 and 1%, two-tailed), while heterogeneous data were analyzed by the Aspin-Welch t-test (levels of significance: 5 and 1%, two-tailed)

Clinical signs:

no effects observed

Description (incidence and severity):

No deaths or abnormal signs were observed.
Excoriation of 1 male (Nol. 1003) in control group occurred on day 28.

Recovery Period
No abnormalities were observed in any animals throughout the recovery period.

Mortality:

no mortality observed

Description (incidence):

Body weight and weight changes:

no effects observed

Description (incidence and severity):

no significant difference was observed.

During recovery
no significant difference was observed.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

no significant difference was observed.

During recovery
no significant difference was observed.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly high number of white blood cell in males of the 12 mg/kg group, significant high values of large non-stained cell ratio in leukocyte percentage in males of the 12 mg/kg group but significant lower values in females of the 12 mg/kg group, significant high percentage of eosinophils in females of the 300 mg/kg group, and significant high number of lymphocytes in white blood cells, of Basophils and of large non-stained cells in male of the 12 mg/kg group, a significant high number of eosinophils in females of the 300 mg/kg group were recognized.

End of Recovery Period
There was no significant difference in comparison between the control group and any test items in males and females at the dose of 300 mg / kg

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly high total cholesterol levels were observed in females at the dose of 300 mg/kg.

End of Recovery Period:
Significant low values of glucose in males of the 300 mg/kg group, significant high values of glucose in female of the 300 mg/kg group, and significantly low values of AST, LDH and A/G ratio in females of the 300 mg/kg group were recognized.

Urinalysis findings:

no effects observed

Description (incidence and severity):

From qualitative aspect, ketone body positive was 3/12 males in the control group (>7.6 mg/dL), 2/6 males in the 60 mg/kg administration group, all males and 11/12 females in the 300 mg/kg administration group, an increase tendency of the ketone body positive in males and females of the 300 mg/kg administration group.
No other abnormalities were found in any other animals, and no significant differences were found in urine volume, water intake and urine osmotic pressure between the control group and the sex of each test substance administered group.

During recovery
No abnormalities were found in qualitative items in any of the animals, and no significant difference was found in urine volume, water intake and urine osmotic pressure between the control group and both sexes of 300 mg / kg administration group.

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):

Liver: Significantly high absolute and relative weights were observed in males and females at the dose of 300 mg/kg.

Kidney: A significant high value of absolute weight was found in male of 60 mg /kg and more group, and a significant high value of relative weight was observed in male of 300 mg/kg group.

End of Recovery Period:
Epididymis: A significant high value of relative weight was observed in the 300 mg/kg administration group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Thyroid gland: small size was observed in 1/6 males of the 12 mg/kg group.
Stomach: thickening wall of forestomach was observed in 2/6 males and 5/6 females in the 300 mg/kg group.
Testes: small size was observed in 1/6 males of the 300 mg/kg group.
Epididymis: small size was observed in 1/6 males of the 300 mg/kg group.
Other: excoriation of 1/6 male in control group occurred.

End of Recovery Period:
No abnormality was found in any of the animals.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes suspected to be caused by the administration of the test substance were observed in the stomachs of male and female, the kidneys and liver of male. Changes in the spleen were considered to be due to administration of the test substance.
Stomach: The following findings at necropsy were observed including 2/6 males and 5/6 females in the 300 mg/kg group: wall thickening of the forestomach. Minor or mild hyperplasia of pavement epithelium of forestomach in all females and in 5/6 males of 300 mg/kg group, and minor or mild hyperplasia of pavement epithelium of the boundary edge in 2/6 males and in 4/6 females of the 300 mg/kg group.
Kidney: Increasing tendency of minor or mild eosinophilic body of tubular cells in 1/6 males of the control group, 3/6 males of the 60 mg/kg group, and all males of the 300 mg/kg group, and males in more than 60 mg/kg group was observed.
Liver: Minor centrilobular hepatocyte hypertrophy was observed in 4/6 males of the 300 mg/kg group.
Spleen: Minor extramedullary hematopoiesis was recognized in 1/6 females of control group, 3/6 male and 2/6 female of 12mg/kg group, 3/6 for both sexes of the 60 mg/kg group, 2/6 male and 3/ females of the 300 mg/kg group.

End of Recovery Period:
Changes thought to be due to administration of the test substance were observed in the male’s kidney.
Kidney: Increasing tendency of minor eosinophilic body of tubular cells in 1/6 males of the control group, 3/6 males of the 300 mg/kg group was observed.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

Detailed Clinical Observation, Manipulative Test, Grip Strength and Motor Activity:

Detailed Clinical Observation
During administration:
Low number of standingup was observed in female of 300 mg / kg administration group at 1 week administration of the test substance. In addition, mild slobber was observed in males of the 300 mg/kg administration group, 2/12 in week 2 and 4/12 in week 3.

During recovery:
There was no abnormality in any of the test items, and no significant difference was found between the control group and the 300 mg / kg administration group in both males and females.

Manipulative Test
During administration:
There was no abnormality in any of the test items, and no significant difference was observed between the control group and the test substance administration group in both males and females.

During recovery:
There was no abnormality in any of the test items, and no significant difference was observed between the control group and the 300 mg/kg administration group in both males and females.

Grip Strength
During administration:
There was no significant difference between the control group and each test substance-administered group in both sexes.

During recovery:
There was no significant difference in the listening between the control group and the 300 mg/kg administration group.

Motor Activity
During administration:
no significant difference was observed.

During recovery:
no significant difference was observed.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

As a result, the NOAEL of 4-phenylbutenone under the test condition was estimated to be 60 mg/kg/day from histopathological examination of the liver and stomach as the main change in both sexes.

Executive summary:

A 28d repeated oral administration toxicity test of 4-phenylbutenone to 6-week old Sprague-Dawley SPF rats (Crl: CD (SD), each group 6 or 12 males or females) via gavage was carried out. Dose is 0 (vehicle 0.5 w/v% aqueous methyl cellulose solution: control group), 12, 60 and 300 mg/kg, and control group as well as 300 mg/kg groups (6 males and 6 females in each group), 2 weeks’ recovery period. Reversibility of toxicity change was examined.

For general clinical observation, no effect related to administration of the test substance was observed.

For detailed clinical observation, manipulative test, grip strength and motor activity,low number of standingup was observed in female of 300 mg/kg administration group at 1 week administration of the test substance. In addition, mild slobber was observed in males of the 300 mg/kg administration group.

For body weight and food intake: no effect related to administration of test substance.

For urinalysis (including water intake): no effect related to administration of test substance.

Hematology examination: no effect related to test substance administration.

Blood chemistry analysis: high value of total cholesterol was observed in females of 300 mg/kg administration group.

Pathological examination: absolute and relative liver weight in male and female of

300 mg/kg administration group is high, hypertrophy of centrilobular hepatocyte in the liver was also observed in 300 mg/kg male group in histological examination.

In addition, in male and female 300 mg/kg group, the thickening of the wall of the forestomach was observed macroscopically, and histologically hyperplasia of pavement epithelium of forestomach and of the boundary edge was observed.

Changes seen during the administration period or at the end of the administration period are all recoverable after administration stopped.

As a result, the NOAEL of 4-phenylbutenone under the test condition was estimated to be 60 mg/kg/day from histopathological examination of the liver and stomach as the main change in both sexes.

Reference 3

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.
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
Species:: mouse
Strain:: B6C3F1
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. Mice were quarantined for 14 (males) or 15 (females) days and were 6 to 7 weeks old on the first day of the study.
- Housing: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: 14 / 15 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
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 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.
Duration of treatment / exposure:: 14 weeks
Frequency of treatment:: ad libitum, available in diet for 14 weeks
Dose / conc.:: 0.025 other: %
Remarks:: approx. 50 and 55 mg/kg bw in females and males, respectively
Basis:
nominal in diet
Dose / conc.:: 0.05 other: %
Remarks:: approx. 100 and 110 mg/kg bw in females and males, respectively
Basis:
nominal in diet
Dose / conc.:: 0.1 other: %
Remarks:: approx. 200 and 220 mg/kg bw in females and males, respectively
Basis:
nominal in diet
Dose / conc.:: 0.2 other: %
Remarks:: approx. 350 and 400 mg/kg bw in females and males, respectively
Basis:
nominal in diet
Dose / conc.:: 0.4 other: %
Remarks:: Doses / Concentrations:
approx. 600 and 750 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 (if not random): Animals were distributed randomly into groups of approximately equal initial mean body weights.
Positive control:: none
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: Blood was collected for hematology analyses from surviving mice at study termination.
- 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: on days 4 and 24 and from core study animals at the end of the studies for haematology and clinical chemistry (rats).
- 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.
Clinical signs:: effects observed, treatment-related
Description (incidence and severity):: One male receiving 0.2% and one control female died before the end of the study. Hyperactivity in both sexes was the only clinical finding.
Mortality:: mortality observed, treatment-related
Description (incidence):: One male receiving 0.2% and one control female died before the end of the study. Hyperactivity in both sexes was the only clinical finding.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Mean body weights of males and females receiving 0.4% 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):: leukocytes and the segremented neutrophils and the lymphocytes were decreased in male mice treated with 0.1 and 0.2 % (P = 0.05). In addition, the monocytes were significantly decreased in male mice treated with 0.1, 0.2 and 0.4 % ( P = 0.01)...
Clinical biochemistry findings:: not examined
Urinalysis findings:: not specified
Behaviour (functional findings):: not specified
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: Absolute R. kidney weight was sig.decreased in males treated with 0.4 % (P = 0.01). Relative thymus weight was increased in males treated with 0.4 % ( P = 0.05). Females treated with 0.4 % the absolute heart weight was sig. different.
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: There were significantly increased incidences of olfactory epithelial atrophy of the nose in males and females receiving 0.4%.
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Histopathological findings: neoplastic:: not examined
Details on results:: CLINICAL SIGNS AND MORTALITY
One male receiving 0.2% and one control female died before the end of the study. Hyperactivity in both sexes was the only clinical finding.

BODY WEIGHT AND WEIGHT GAIN
Mean body weights of males and females receiving 0.4% 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
The mean cell haemoglobin was significantly (P = 0.01) increased in male mice treated with 0.025 % and 0.1 %.
Moreover, the leukocytes and the segrmented neutrophils and the lymphocytes were decreased in male mice treated with 0.1 and 0.2 % (P = 0.05).
In addition, the monocytes were significantly decreased in male mice treated with 0.1, 0.2 and 0.4 % ( P = 0.01).
In female mice, the erythrocyte count was significantly decreased in animals treated with 0.05 % (P = 0.05). The mean cell volume was significantly incresed in females treated with 0.025 % (P = 0.05).

ORGAN WEIGHTS
The absolute R. kidney weight was significantlydecreased in males treated with 0.4 % (P = 0.01). Moreover, the relative thymus weight was increased in males treated with 0.4 % ( P = 0.05).
In females treated with 0.4 % the absolute heart weight was significantly different ( P = 0.01). In addition, the absolute liver weight was increased in females treated with 0.025 and 0.05 % ( P = 0.01).

HISTOPATHOLOGY: NON-NEOPLASTIC
There were significantly increased incidences of olfactory epithelial atrophy of the nose in males and females receiving 0.4%.

OTHER FINDINGS
Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male mice; however, it exhibits potential for reproductive toxicity in female mice based upon an increased probability of extended diestrus at the lowest and the highest exposure concentrations.
: Key result
Dose descriptor:: NOAEL
Effect level:: 350 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: female
Basis for effect level:: histopathology: non-neoplastic
: Key result
Dose descriptor:: NOAEL
Effect level:: 400 mg/kg bw/day (nominal)
Based on:: test mat.
Sex:: male
Basis for effect level:: histopathology: non-neoplastic
Critical effects observed:: not specified
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 only hyperactivity in both sexes. An NOAEL was derived (350 (females, and 400 (males) mg/kg bw).
Executive summary:: The substance methyl trans-styryl ketone was investigated for its repeated dose toxicity via the oral route in mice (NTP, 2011). Groups of 10 male and 10 female mice 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 55, 110, 220, 400, or 750 mg/kg to males and 50, 100, 200, 350, or 600 mg/kg to females) for 14 weeks. There were no treatment-related deaths in either sex (one male receiving 0.2% died following blood collection, and one control female was found dead). Mean body weights of males and females receiving 0.4% were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Hyperactivity in both sexes was the only treatment-related clinical finding. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male mice; however, it exhibits potential for reproductive toxicity in female mice based upon an increased probability of extended diestrus at the lowest and the highest exposure concentrations. There were significantly increased incidences of olfactory epithelial atrophy of the nose in males and females receiving 0.4%. An NOAEL was derived (350 (females), and 400 (males) mg/kg bw).

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 145 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: A 90-day repeated dose toxicity studies in rats is available for benzalacetone. This study is used for the DNEL derivation and further risk assessment (well-documented NTP report).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

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.
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
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
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
Dose / conc.:: 22 mg/kg bw/day (nominal)
Dose / conc.:: 44 mg/kg bw/day (nominal)
Dose / conc.:: 87.5 mg/kg bw/day (nominal)
Dose / conc.:: 175 mg/kg bw/day (nominal)
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
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.
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.
: 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
Critical effects observed:: not specified
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.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 87.5 mg/kg bw/day
Study duration:: subchronic
Species:: rat
Quality of whole database:: A 90-day repeated dose toxicity studies in rats is available for benzalacetone. This study is used for the DNEL derivation and further risk assessment (well-documented NTP report).

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

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.
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
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
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
Dose / conc.:: 22 mg/kg bw/day (nominal)
Dose / conc.:: 44 mg/kg bw/day (nominal)
Dose / conc.:: 87.5 mg/kg bw/day (nominal)
Dose / conc.:: 175 mg/kg bw/day (nominal)
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
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.
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.
: 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
Critical effects observed:: not specified
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.

Endpoint conclusion

Endpoint conclusion:: adverse effect observed
Dose descriptor:: NOAEL
Study duration:: subchronic
Species:: rat
Quality of whole database:: A 90-day repeated dose toxicity studies in rats is available for benzalacetone. This study is used for the DNEL derivation and further risk assessment (well-documented NTP report).

Additional information

Repeated dose oral:

A 28d repeated oral administration toxicity test ofBenzalacetone (CAS 1896 -62 -4) to 6-week old Sprague-Dawley SPF rats (Crl: CD (SD), each group 6 or 12 males or females) via gavage was carried out according to international acceptable method by the Japanes Minitry of Health, Labour and Welfare . Dose is 0 (vehicle 0.5 w/v% aqueous methyl cellulose solution: control group), 12, 60 and 300 mg/kg, and control group as well as 300 mg/kg groups (6 males and 6 females in each group), 2 weeks’ recovery period. Reversibility of toxicity change was examined. For general clinical observation, no effect related to administration of the test substance was observed. For detailed clinical observation, manipulative test, grip strength and motor activity,low number of standingup was observed in female of 300 mg/kg administration group at 1 week administration of the test substance. In addition, mild slobber was observed in males of the 300 mg/kg administration group. For body weight and food intake: no effect related to administration of test substance. For urinalysis (including water intake): no effect related to administration of test substance. Hematology examination: no effect related to test substance administration. Blood chemistry analysis: high value of total cholesterol was observed in females of 300 mg/kg administration group. Pathological examination: absolute and relative liver weight in male and female of 300 mg/kg administration group is high, hypertrophy of centrilobular hepatocyte in the liver was also observed in 300 mg/kg male group in histological examination. In addition, in male and female 300 mg/kg group, the thickening of the wall of the forestomach was observed macroscopically, and histologically hyperplasia of pavement epithelium of forestomach and of the boundary edge was observed.

Changes seen during the administration period or at the end of the administration period are all recoverable after administration stopped.

As a result, the NOAEL was estimated to be 60 mg/kg/day from histopathological examination of the liver and stomach as the main change in both sexes.

Benzalacetone (CAS 1896 -62 -4) was investigated for its repeated dose toxicity (90 days) via the oral route in rats (NTP, 2012a). 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%. A NOAEL was derived (145 and 150 mg/kg bw for female and male rats, respectively).

Benzalacetone (CAS 1896 -62 -4) was investigated for its repeated dose toxicity via the oral route in mice (NTP, 2012a). Groups of 10 male and 10 female mice 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 55, 110, 220, 400, or 750 mg/kg to males and 50, 100, 200, 350, or 600 mg/kg to females) for 14 weeks. There were no treatment-related deaths in either sex (one male receiving 0.2% died following blood collection, and one control female was found dead). Mean body weights of males and females receiving 0.4% were significantly less than those of the controls. Feed consumption by exposed groups was similar to that by the controls. Hyperactivity in both sexes was the only treatment-related clinical finding. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male mice; however, it exhibits potential for reproductive toxicity in female mice based upon an increased probability of extended diestrus at the lowest and the highest exposure concentrations. There were significantly increased incidences of olfactory epithelial atrophy of the nose in males and females receiving 0.4%. A NOAEL was derived (350 and 400 mg/kg bw for female and male mice, respectively).

Repeated dose dermal:

Benzalacetone (CAS 1896 -62 -4) was investigated for its repeated dose toxicity via the dermal route in rats (NTP, 2012a). 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.

Benzalacetone (CAS 1896 -62 -4) was investigated for its repeated dose toxicity via the dermal route in mice (NTP, 2012a). Groups of 10 male and 10 female mice were dermally administered 0, 87.5, 175, 350, 700, or 1,400 mg methyl trans-styryl ketone/kg body weight in 95% ethanol, 5 days per week for 13 weeks. All mice in the 700 and 1,400 mg/kg groups were sacrificed moribund before the end of the study. The final mean body weights of surviving groups of dosed males and females were similar to those of the vehicle controls; however, the mean body weight gains of the 175 mg/kg groups were significantly less than those of the vehicle controls. Clinical findings at the site of application included dermal irritation in 350 mg/kg males and crust formation in all 700 and 1,400 mg/kg mice except one female. Results of sperm motility and vaginal cytology evaluations indicated methyl trans-styryl ketone is unlikely to be a reproductive toxicant in male or female mice at the doses used in this study. There were treatment-related increased incidences of epidermal hyperplasia, hyper-keratosis, chronic active inflammation, epidermal necrosis, sebaceous gland hypertrophy, and hair follicle hyperplasia in the skin at the site of application in males and females. There were increased incidences of olfactory epithelial atrophy of the nose in groups of males and females administered 350 mg/kg or greater. A LOAEL was derived (87.5 mg/kg bw).

Justification for classification or non-classification

According to the Regulation (EC) No 1272/2008 the test material does not meet the criteria for classification and will not require labelling.

Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.

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 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 (10⁶/testis)	179.63 ± 7.44	179.06 ± 9.84	177.50 ± 5.51	190.50 ± 7.21
Spermatid heads (10³/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 (10⁶/cauda epididymis)	82.8 ± 5.8	66.9 ± 9.6	59.6 ± 9.2	62.2±8.6
Sperm (10³/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

TABLE 2 Survival, Body Weights, and Feed Consumption of Mice in the 3-Month Feed Study of Methyl trans-Styryl Ketone'
					Final Weight
		Initial	Final	Change in	Relative	Feed	Feed
Concentration		Body Weight	Body Weight Body Weight		to Controls	Consumption	Consumption
	Survival^b	(g)	(g)	(g)	(%)	Week 1^c	Week14
Male
0	10/10	20.4±0.3	31.3±0.6	10.9 ± 0.5		5.2	5.3
0.025	10/10	20.3±0.3	31.4±1.1	11.1 ± 0.9	100	6.3	6.1
0.05	10/10	20.0 ± 0.3	30.2 ± 0.5	10.2 ± 0.3	96	5.0	5.5
0.1	10/10	20.4 ± 0.4	31.3±0.7	11.0±0.5	100	4.7	5.7
0.2	10/10	20.5±0.3	31.0±0.8	10.5 ± 0.7	99	4.2	5.5
0.4	10/10	20.2±0.5	28.5±0.6*	8.3 ± 0.6*	91	3.6	4.6
Female
0	9/10^d	16.4±0.3	22.7 ± 0.5	6.2 ± 0.3		3.2	3.9
0.025	10/10	17.2±0.3	24.9 ± 0.4	7.7 ± 0.2	110	3.6	3.9
0.05	10/10	17.5±0.4	25.4 ± 0.7	7.9 ± 0.5	112	3.4	4.2
0.1	10/10	16.8±0.3	23.5 ± 0.4	6.7 ± 0.4	104	3.3	4.5
0.2	10/10	16.9±0.3	22.0 ± 0.3	5.1 ± 0.4	97	3.2	3.6
0.4	10/10	16.7±0.3	20.7 ± 0.4**	4.0 ± 0.4**	92	2.1	3.1
* Significantly different (P<0.05) from the control group by Dunnett's test
** Significantly different (P<0.01) from the control group by Williams' test
^a Weights and weight changes are given as mean ± standard error. Feed consumption is expressed as grams per animal per day. Subsequent calculations are based on animals surviving to the end of the study.
^b Number of animals surviving at 14 weeks/number initially in group
^c Except week 2 for 0% males
^d Week of death: 12

Table 4
Haematology Data for Mice in the 3-Month Feed Study of Methyl trans-Styryl Ketone^a
	0%	0.025%	0.05%	0.1%	0.2%	0.4%
Male
n	10	10	10	10	10	10
Hematocrit (%)	50.7 ± 1.0	50.6 ± 0.7	50.6 ± 1.0	50.7 ± 1.1	48.5±0.9	49.2 ± 0.9
Hemoglobin (g/dL)	16.1 ± 0.3	16.2 ± 0.2	16.4 ± 0.5	16.3±0.3	15.5±0.3	15.7 ± 0.3
Erythrocytes (10⁶/uL)	10.23±0.20	10.06 ± 0.14	10.11 ± 0.23	10.08±0.20	9.74 ± 0.17	9.88 ± 0.15
Reticulocytes (10⁶/uL)	0.34 ± 0.02	0.32 ± 0.02	0.31 ± 0.02	0.34 ± 0.03	0.32 ± 0.03	0.31 ± 0.02
Mean cell volume (fL)	49.5 ± 0.2	50.4 ± 0.2	50.3±0.4	50.2 ± 0.2	49.9 ± 0.2	49.7 ± 0.2
Mean cell hemoglobin (pg)	15.8±0.1	16.1 ± 0.1**	16.1 ± 0.2	16.2 ± 0.1**	15.9 ± 0.1	15.8 ± 0.0
Mean cell hemoglobin concentration
(g/dL)	31.8±0.1	32.0 ± 0.2	32.0 ± 0.3	32.2 ± 0.1	31.9 ± 0.1	31.9 ± 0.1
Platelets (10³/uL)	588.0 ± 60.1	528.2 ± 42.4	583.4 ± 41.5	548.9 ± 30.5	572.8±61.2^b	582.6 ± 33.6
Leukocytes (10³/uL)	6.15±0.57	4.56 ± 0.84	4.95 ± 0.72	3.35 ± 0.38*	3.55 ± 0.45*	4.05 ± 0.51
Segmented neutrophils (10³/uL)	0.67 ± 0.10	0.56 ± 0.17	0.35 ± 0.06	0.26 ± 0.04**	0.35 ± 0.07*	0.38 ± 0.08
Lymphocytes (10³/uL)	5.19 ± 0.49	3.86 ± 0.70	4.41 ± 0.63	2.95 ± 0.33*	3.07 ± 0.36*	3.53 ± 0.46
Monocytes (10³/uL)	0.21 ± 0.03	0.11 ± 0.02	0.15±0.04	0.09 ± 0.01**	0.09 ± 0.02**	0.10 ± 0.02**
Basophils (10³/uL)	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 (10³/uL)	0.09 ± 0.02	0.03 ± 0.01	0.05 ± 0.02	0.05 ± 0.02	0.04 ± 0.01	0.04 ± 0.02
Female
n	9	10	10	10	10	10
Hematocrit (%)	50.0 ± 0.5	49.0 ± 0.9	48.2 ± 0.5	49.9 ± 0.9	50.1 ± 0.4	49.1 ± 0.7
Hemoglobin (g/dL)	17.0 ± 0.1	16.5±0.3	16.5±0.2	16.9 ± 0.3	16.8 ± 0.1	16.6 ± 0.2
Erythrocytes (10⁶/uL)	9.74 ± 0.10	9.41 ± 0.17	9.26 ± 0.09*	9.66 ± 0.16	9.71 ± 0.07	9.62 ± 0.13
Reticulocytes (10⁶/uL)	0.35 ± 0.02	0.39 ± 0.03	0.38 ± 0.02	0.37 ± 0.03	0.34 ± 0.02	0.41 ± 0.03
Mean cell volume (fL)	51.4 ± 0.2	52.2 ± 0.1*	52.1 ± 0.2	51.8±0.3	51.7 ± 0.2	51.1 ± 0.2
Mean cell hemoglobin (pg)	17.5 ± 0.1	17.5 ± 0.1	17.8 ± 0.1	17.5 ± 0.1	17.3 ± 0.1	17.3 ± 0.1
Mean cell hemoglobin concentration
(g/dL)	34.0 ± 0.2	33.6 ± 0.2	34.1 ± 0.1	33.8 ± 0.2	33.5±0.2	33.8 ± 0.2
Platelets (10³/uL)	816.6 ± 53.2	833.9 ± 39.2	890.0 ± 37.2	786.4 ± 68.0	858.9 ± 27.8	817.8 ± 25.7
Leukocytes (10³/uL)	5.79 ± 0.48	4.46 ± 0.25	4.40 ± 0.22	5.21 ± 0.48	6.01 ± 0.41	5.88 ± 0.41
Segmented neutrophils (10³/uL)	0.76 ± 0.17	0.50 ± 0.03	0.49 ± 0.06	0.61 ± 0.08	0.69 ± 0.09	0.71 ± 0.07
Lymphocytes (10³/uL)	4.81 ± 0.35	3.77 ± 0.25	3.73 ± 0.20	4.38 ± 0.40	5.03 ± 0.34	4.93 ± 0.35
Monocytes (10³/uL)	0.18±0.03	0.13±0.02	0.15±0.02	0.18±0.04	0.20 ± 0.04	0.18 ± 0.04
Basophils (10³/uL)	0.007 ± 0.007	0.003 ± 0.003	0.006 ± 0.006	0.008 ± 0.006	0.008 ± 0.008	0.013 ± 0.009
Eosinophils (10³/uL)	0.04 ± 0.03	0.06 ± 0.01	0.04 ± 0.02	0.04 ± 0.02	0.09 ± 0.03	0.05 ± 0.03
* Significantly different (P<0.05) from the control group by Dunn's test
** Significantly different (P<0.01) from the control group by Dunn's or Shirley's test
^a Data are presented as mean ± standard error. Statistical tests were performed on unrounded data.
^bn=9

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 (10⁶/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 (10⁶/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 (10³/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 (10³/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 (10³/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 (10³/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 (10³/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 (10³/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 (10³/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 (10⁶/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 (10⁶/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 (10³/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 (10³/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 (10³/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 (10³/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 (10³/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 (10³/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 (10³/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 3 Incidences of Non-neoplastic Lesions of the Nose in Mice in the 3-Month Feed Study of Methyl trans-Styryl Ketone
		0.025%	0.05%	0.1%	0.2%	0.4%
Male
Number Examined Microscopically	10	10	10	10	10	10
Olfactory Epithelium, Atrophy	0	0	0	0	1 (1.0)^b	10**	(1.0)
Female
Number Examined Microscopically	9	10	10	10	10	10
Olfactory Epithelium, Atrophy	0	0	0	0	0	10**	(1.0)
** Significantly different (P<0.01) from the control group by the Fisher exact test
^a Number of animals with lesion
^b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

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
	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 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

Registration Dossier

Registration Dossier

Diss Factsheets

4-phenylbutenone

Endpoint summary

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Reference 3

Endpoint conclusion

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint conclusion

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint conclusion

Additional information

Repeated dose oral:

Repeated dose dermal:

Justification for classification or non-classification

Referenceopen all close all