Registration Dossier

Administrative data

Description of key information

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This information comes from an NTP-report (TR 572), which deals with the related substance: methyl trans-styryl ketone. The quality of this report is considered to be high.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity 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
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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
Remarks:
Doses / Concentrations:
0.025 % (approx. 18 and 19 mg/kg bw in females and males, respectively)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.05 % (approx. 36 and 38mg/kg bw in females and males, respectively)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.1 % (approx. 72 and 77 mg/kg bw in females and males, respectively)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.2 % (approx. 145 and 150 mg/kg bw in females and males, respectively)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.4 % (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).
Dose descriptor:
NOAEL
Effect level:
145 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Critical effects observed:
not specified
Table 2 Survival, Body Weights, and Feed Consumption of Rats in the 3-Month Feed Study of Methyl trans-Styryl Ketonea
Concentration(%) Survivalb 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            
KidneyaNephropathy*5 10 6 (1.0)c 10 8 (1.0) 108 (1.0) 109 (1.0) 10 10* (1.0) 10 10* (1.6)
Nose Respiratory Epithelium, Hyperplasia, Goblet Cell 10 0 10 4* (1.0)c 103 (1.0) 10 4* (1.3) 103 (1.0) 10 9** (1.3)
Female
Nose Respiratory Epithelium, Hyperplasia, Goblet Cell 10 3 (1.0) 10 0 103 (1.0) 103 (1.0) 103 (1.0) 10 7 (1.0)
*  Significantly different (P<0.05) from the control group by the Fisher exact test
** P<0.01
a  Number of animals with tissue examined microscopically
b  Number of animals with lesion
c  Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Table 4 Haematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study of Methyl trans-Styryl Ketonea
  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± 0.10b
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 ± 0.25b
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 ± 0.22b
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 ± 0.000b
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 ± 0.01b
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 ± 1.73b 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 ± 0.23b 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 ± 1.40b 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 ± 0.07b 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± 0.000b 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 ± 0.05b 0.02 ± 0.01 0.07 ± 0.03 0.10 ± 0.02 0.04 ± 0.02 0.05 ± 0.02
Clinical Chemistry
Day 4 10 10 10 10 10 9
Day 24 10 10 10 10 10 9
Week 14 10 10 10 10 10 10
Urea nitrogen (mg/dL)
Day 4 12.5 ± 0.5 14.0 ± 0.6 12.9 ± 0.4 12.8±0.6 13.1 ± 0.4 14.7 ± 0.7
Day 24 16.9 ± 0.5 16.0 ± 0.7 17.6 ± 0.5 16.7 ± 0.5 15.4 ± 0.5 19.1 ± 0.7
Week 14 17.5 ± 0.3 17.2 ± 0.5 17.3 ± 0.4 17.1 ± 0.3 16.6 ± 0.5 17.0 ± 0.4
Creatinine (mg/dL)
Day 4 0.42 ± 0.01 0.39 ± 0.02 0.40 ± 0.01 0.41 ± 0.02 0.42 ± 0.01 0.47 ± 0.02
Day 24 0.41 ± 0.01 0.40 ± 0.00 0.40 ± 0.01 0.41 ± 0.01 0.41 ± 0.01 0.39 ± 0.02
Week 14 0.56±0.02 0.55 ± 0.02 0.58 ± 0.02 0.53 ± 0.02 0.57 ± 0.02 0.56 ± 0.02
Total protein (g/dL)
Day 4 6.2 ± 0.1 6.1 ± 0.1 6.1 ± 0.1 6.2 ± 0.1 6.2 ± 0.1 6.2 ± 0.1
Day 24 6.7 ± 0.1 6.5 ± 0.1 6.5 ± 0.1 6.7 ± 0.1 6.6 ± 0.0 6.6 ± 0.1
Week 14 7.2 ± 0.1 7.3 ± 0.1 7.2 ± 0.1 7.2 ± 0.1 7.3 ± 0.1 7.3 ± 0.1
Albumin (g/dL)
Day 4 4.4 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.1
Day 24 4.6±0.0 4.6 ± 0.0 4.5 ± 0.0 4.6 ± 0.0 4.5 ± 0.1 4.6 ± 0.1
Week 14 5.0±0.1 5.1 ± 0.1 5.0 ± 0.1 5.0 ± 0.1 5.0 ± 0.1 5.0 ± 0.1
Globulin (g/dL)
Day 4 1.8±0.1 1.8 ± 0.0 1.8 ± 0.1 1.8 ± 0.0 1.9 ± 0.1 1.9 ± 0.1
Day 24 2.1 ± 0.0 2.0 ± 0.0 2.0 ± 0.1 2.0 ± 0.0 2.1 ± 0.1 2.0 ± 0.0
Week 14 2.2 ± 0.1 2.2 ± 0.1 2.2 ± 0.1 2.2 ± 0.1 2.3 ± 0.1 2.2 ± 0.1
Albumin/globulin ratio
Day 4 2.4 ± 0.1 2.4 ± 0.1 2.4 ± 0.1 2.4 ± 0.1 2.3 ± 0.1 2.3 ± 0.1
Day 24 2.2 ± 0.0 2.3 ± 0.0 2.2 ± 0.1 2.3 ± 0.0 2.2 ± 0.1 2.2 ± 0.0
Week 14 2.3 ± 0.1 2.3 ± 0.1 2.3 ± 0.1 2.3 ± 0.1 2.2 ± 0.1 2.2 ± 0.0
Alanine aminotransferase (IU/L)
Day 4 47 ± 1 50 ± 1 48 ± 1 49 ± 1 50 ± 2 52 ± 1*
Day 24 35 ± 1 34 ± 1 32 ± 1 32 ± 1 35 ± 1 32 ± 1
Week 14 85 ± 10 67 ± 5 69 ± 10 61 ± 5 68 ± 8 49 ± 3**
Alkaline phosphatase (IU/L)
Day 4 583 ± 13 565 ± 16 580 ± 11 582 ± 15 580 ± 19 587 ± 11
Day 24 370 ± 9 357 ± 7 355 ± 6 349 ± 11 374 ± 7 353 ± 3
Week 14 180±8 190 ± 5 184 ± 4 189 ± 3 182 ± 3 182 ± 6
Creatine kinase (IU/L)
Day 4 423 ± 59 435 ± 63 395 ± 58 420 ± 79 488 ± 88 593 ± 115
Day 24 258 ± 32 295 ± 36 280 ± 30 304 ± 47 332 ± 27 307 ± 26
Week 14 278 ± 39 200 ± 21 231 ± 34 199 ± 25 182 ± 13 199 ± 15
Sorbitol dehydrogenase (IU/L)
Day 4 15±1 15 ± 1 18 ± 2 13 ± 1 12 ± 1 14 ± 1
Day 24 22 ± 1 19 ± 1 21 ± 2 22 ± 2 20 ± 2 19 ± 1
Week 14 31 ± 2 31 ± 2 32 ± 1 32 ± 1 33 ± 2 29 ± 1
Bile salts (umol/L)
Day 4 15.9 ± 1.3 22.9 ± 2.0* 23.2 ± 1.3* 23.7 ± 2.0* 20.6 ± 2.2 23.5 ± 2.4*
Day 24 17.7 ± 1.5 20.9 ± 1.9 22.0 ± 2.4 22.9 ± 1.5 18.7 ± 1.8 19.3 ± 2.4
Week 14 36.7 ± 4.0 39.1 ± 3.1 28.6 ± 1.0 45.7 ± 3.1 30.4 ± 2.8 29.7 ± 3.4
*  Significantly different (P<0.05) from the control group by Dunn's or Shirley's test
**P<0.01
a  Data are presented as mean ± standard error. Statistical tests were performed on unrounded data.
bn=9

Table 5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Feed Study of Methyltrans-Styryl Ketonea
  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 ± 0.09b 1.39 ± 0.07 1.38±0.04 1.41 ± 0.04b 1.44 ± 0.06
Relative 4.354 ± 0.129 4.600 ± 0.243b 4.196±0.198 4.184±0.099 4.331 ± 0.146b 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± 3c 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 Ketonea
  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 Ketonea
  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 femalesb 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
Conclusions:
The study was performed with the related 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 NOEAL was derived (145 (females, and 150 (males) mg/kg bw).
Executive summary:

The related 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 NOEAL was derived (145 (females, and 150 (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:
No repeated dose toxicity studies are available for benzalacetone. Therefore, a subchronic study in rats conducted with the related substance methyl-trans-styryl ketone, the nearest analogue, 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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This information comes from an NTP-report (TR 572), which deals with the related substance: methyl trans-styryl ketone. The quality of this report is considered to be high.
Qualifier:
equivalent or similar to
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
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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
Remarks:
Doses / Concentrations:
22 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
44 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
87.5 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
175 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
350 mg/ kg bw/ day
Basis:
nominal per unit body weight
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.
Dose descriptor:
NOAEL
Effect level:
87.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
TABLE 2
Survival and Body Weights of Rats in the 3-Month Dermal Study of Methyl trans-Styryl Ketonea
Dose (mg/kg) Survivalb initial Body Weight (g) Final Body Weight (g) Change in Body Weight (g) Final Weight Relative to Controls(%)
Male          
0 10/10 100±2 309 ± 6 209 ± 5
22 10/10 100±3 311 ± 7 211 ± 6 100
44 10/10 100 ± 3 308 ± 8 208 ± 6 99
87.5 10/10 101 ± 3 300 ± 8 200 ± 5 97
175 10/10 99±3 284±6* 185 ± 4** 92
350 10/10 99±3 278±7** 179 ± 5** 90
Female
0 10/10 94 ± 1 180 ± 4 86 ± 3
22 10/10 93 ± 2 180 ± 4 87 ± 3 100
44 10/10 93 ± 2 183 ± 4 90 ± 3 102
87.5 10/10 94 ± 2 174 ± 2 80 ± 2 97
175 10/10 93 ± 2 175 ± 3 83 ± 3 98
350 10/10 94 ± 1 176 ± 2 82 ± 3 98
*  Significantly different (P<0.05) from the vehicle control group by William's test
** P<0.01
a  Weights and weight changes are given as mean ± standard error.
b  Number of animals surviving at 14 weeks/number initially in group

TABLE 3
Incidences of Selected Nonneoplastic Lesions in Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketone
  Vehicle Control 22 mg/kg 44 mg/kg 87.5 mg/kg 175 mg/kg 350 mg/kg
Male
Nosea 10 10 10 10 10 10
Respiratory Epithelium,
Hyperplasia, Goblet Cellb 2 (1.0)c 6 (1.3) 1 (1.0) 6 (1.2) 4 (1.3) 9** (1.7)
Skin, Site of Application 10 10 9 10 10 10
Sebaceous Gland,
Hypertrophy 0 1 (1.0) 3 (1.0) 6** (1.2) 10** (1.8) 7** (1.9)
Epidermis, Hyperplasia 0 3 (1.0) 6** (1.0) 8** (1.5) 10** (2.2) 10** (2.3)
Hyperkeratosis 1 (1.0) 6* (1.2) 7** (1.4) 10** (1.7) 10** (1.9) 9** (2.1)
Inflammation, Chronic Active 0 0 0 0 3 (1.7) 4*(2.5)
Necrosis 0 0 0 0 3 (2.0) 3 (2.3)
Female
Nose 10 10 10 10 10 10
Respiratory Epithelium,
Hyperplasia, Goblet Cell 0 8** (1.0) 3 (1.0) 3 (1.0) 7** (1.4) 5*(1.2)
Skin, Site of Application 10 10 10 10 10 10
Sebaceous Gland, Hypertrophy                         0                     1   (1.0)          5*  (1.0)          7** (1.0)          4*  (1.5)          8** (1.6)
Epidermis, Degeneration 0 0 0 0 2 (3.0) 1 (1.0)
Epidermis, Hyperplasia 0 3 (1.0) 3 (1.0) 7** (1.6) 9** (2.2) 10** (2.2)
Hyperkeratosis 0 6** (1.0) 6** (1.0) 9** (1.9) 8** (1.6) 9** (2.0)
Inflammation, Chronic Active 0 0 0 1 (1.0) 6**(2.5) 4* (2.8)
Necrosis 0 0 0 0 4* (2.0)
Ulcer 0 0 0 0 1 1
*  Significantly different (P<0.05) from the vehicle control group by the Fisher exact test
** P<0.01
a  Number of animals with tissue examined microscopically
b  Number of animals with lesion
c  Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

TABLE 4 Haematology and Clinical Chemistry Data for Rats in the 3-Month Dermal Study of Methyl trans-Styryl Ketonea
  Vehicle Control 22 mg/kg 44 mg/kg 87.5 mg/kg 175 mg/kg 350 mg/kg
Male
Haematology
n Day 4 10 10 10 9 10 10
Day 24 10 10 10 10 10 10
Week14 10 10 10 10 10 10
Haematocrit(%)
Day 4 38.5 ± 0.5 37.5 ± 1.0 38.3±0.3 38.4 ± 0.5 38.7 ± 0.2 38.6 ± 0.4
Day 24 43.8±0.5 43.9 ± 0.7 43.9 ± 0.4 43.7 ± 0.5 43.0 ± 0.3 43.4 ± 0.5
Week14 44.8 ± 0.4 44.1 ± 0.5 44.8 ± 0.4 44.6 ± 0.4 44.6 ± 0.7 44.8 ± 0.5
Haemoglobin (g/dL)
Day 4 13.2 ± 0.2 12.9 ± 0.3 13.2 ± 0.1 13.2 ± 0.1 13.3 ± 0.1 13.2 ± 0.1
Day 24 15.4 ± 0.2 15.5±0.2 15.4 ± 0.1 15.3±0.1 15.0 ± 0.1 15.2 ± 0.2
Week 14 15.6±0.1 15.4 ± 0.1 15.7 ± 0.1 15.6 ± 0.1 15.5±0.1 15.7 ± 0.1
Erythrocytes (106/uL)
Day 4 6.93 ± 0.08 6.78 ± 0.16 6.90 ± 0.07 6.92 ± 0.09 7.04 ± 0.04 7.07 ± 0.07
Day 24 8.02 ± 0.10 8.02 ± 0.11 8.03 ± 0.08 8.00 ± 0.10 7.88 ± 0.05 7.89 ± 0.09
Week 13 8.93 ± 0.06 8.87 ± 0.08 8.99 ± 0.04 8.93 ± 0.07 8.87 ± 0.09 8.98 ± 0.08
Reticulocytes (106/uL)
Day 4 0.59 ± 0.03 0.58 ± 0.03 0.60 ± 0.02 0.58 ± 0.01 0.56 ± 0.02 0.63 ± 0.01
Day 24 0.40±0.04 0.41 ± 0.03 0.41 ± 0.02 0.32 ± 0.01 0.37 ± 0.03 0.38 ± 0.03
Week 14 0.24 ± 0.02 0.19 ± 0.02 0.21 ± 0.02 0.25 ± 0.01 0.22 ± 0.02 0.25 ± 0.01
Mean cell volume (fL)
Day 4 55.4 ± 0.2 55.3 ± 0.3 55.5 ± 0.2 55.2 ± 0.2 54.8 ± 0.3 54.6 ± 0.3
Day 24 54.6±0.2 54.7 ± 0.2 54.7 ± 0.2 54.6 ± 0.2 54.7 ± 0.2 55.0 ± 0.2
Week 14 50.1 ± 0.2 49.7 ± 0.3 49.7 ± 0.4 49.9 ± 0.4 50.3 ± 0.3 49.8 ± 0.1
Mean cell hemoglobin (pg)
Day 4 19.0 ± 0.1 19.1 ± 0.1 19.2 ± 0.1 19.1 ± 0.1 18.9 ± 0.1 18.7 ± 0.1*
Day 24 19.1 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.0 ± 0.0 19.2 ± 0.1
Week 14 17.4 ± 0.1 17.4 ± 0.0 17.5 ± 0.0 17.5 ± 0.1 17.5 ± 0.1 17.5 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4 34.2 ± 0.1 34.4 ± 0.1 34.6 ± 0.1 34.5 ± 0.1 34.4 ± 0.1 34.2 ± 0.1
Day 24 35.0±0.1 35.2 ± 0.1 35.0 ± 0.1 35.0 ± 0.1 34.8 ± 0.1 34.9 ± 0.1
Week 14 34.8±0.2 34.9 ± 0.2 35.1 ± 0.2 35.0 ± 0.2 34.8 ± 0.2 35.0 ± 0.2
Platelets (103/uL)
Day 4 727.3 ± 14.5 706.1 ± 19.1 685.8 ± 35.6 745.6 ± 18.4 743.8 ± 9.6 742.7 ± 19.2
Day 24 563.8 ± 20.0 546.3 ± 23.8 536.1 ± 27.9 552.3 ± 21.6 569.5 ± 11.3 518.0 ± 27.7
Week 14 601.9 ± 10.9 514.0±19.5** 541.2 ± 19.9 535.5 ± 14.4* 546.6 ± 21.6 563.5 ± 16.9
Leukocytes (103/uL)
Day 4 8.72 ± 0.42 8.29 ± 0.56 9.43 ± 0.72 9.67 ± 0.59 10.54 ± 0.77 9.69 ± 0.64
Day 24 12.17 ± 0.54 11.71 ± 0.46 12.48 ± 0.35 12.26 ± 0.41 12.26 ± 0.29 12.06 ± 0.54
Week 14 7.80±0.44 7.62 ± 0.38 8.46 ± 0.31 8.90 ± 0.37 8.28 ± 0.32 7.94 ± 0.44
Segmented neutrophils (103/uL)
Day 4 0.79 ± 0.12 0.89 ± 0.08 0.94 ± 0.14 1.02 ± 0.12 1.48 ± 0.18** 1.95 ± 0.16**
Day 24 1.09 ± 0.14 0.88 ± 0.08 1.30 ± 0.14 1.13±0.17 1.27 ± 0.11 1.24 ± 0.12
Week 14 1.02 ± 0.13 0.98 ± 0.12 1.13±0.10 1.17 ± 0.09 1.16 ± 0.09 1.23 ± 0.12
Lymphocytes (103/uL)
Day 4 7.40±0.33 6.88 ± 0.49 7.89 ± 0.58 8.04 ± 0.51 8.33 ± 0.57 7.19 ± 0.50
Day 24 10.42 ± 0.46 10.05 ± 0.40 10.40 ± 0.33 10.45 ± 0.30 10.10 ± 0.30 10.12 ± 0.43
Week 14 6.48 ± 0.40 6.25 ± 0.32 6.89 ± 0.26 7.17 ± 0.32 6.71 ± 0.35 6.35 ± 0.38
Monocytes (103/uL)
Day 4 0.50±0.06 0.48 ± 0.08 0.58 ± 0.05 0.56 ± 0.08 0.71 ± 0.09 0.50 ± 0.07
Day 24 0.63 ± 0.11 0.72 ± 0.10 0.77 ± 0.09 0.65 ± 0.10 0.83 ± 0.06 0.64 ± 0.11
Week 14 0.22 ± 0.02 0.36 ± 0.07 0.34 ± 0.06 0.38 ± 0.08 0.30 ± 0.04 0.31 ± 0.06
Basophils(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 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 0.04 ± 0.02 0.40 ± 0.02 0.03 ± 0.02 0.05 ± 0.03 0.02 ± 0.01 0.04 ± 0.02
Day 24 0.04 ± 0.02 0.07 ± 0.02 0.01 ± 0.01 0.04 ± 0.02 0.06 ± 0.03 0.06 ± 0.03
Week 14 0.05 ± 0.02 0.02 ± 0.01 0.08 ± 0.03 0.12 ± 0.02 0.06 ± 0.03 0.04 ± 0.01
Clinical Chemistry
n 10 10 10 10 10 10
Ureanitrogen(mg/dL)
Day 4 14.4 ± 0.6 14.6 ± 0.3 13.6 ± 0.6 14.6 ± 0.5 13.9 ± 0.4 14.9 ± 0.5
Day 24 13.1 ± 0.4 12.8 ± 0.4 12.5 ± 0.4 11.7 ± 0.6 12.6 ± 0.5 12.1 ± 0.4
Week 14 16.4 ± 0.5 16.8 ± 0.6 16.7 ± 0.6 17.2 ± 1.1 17.5 ± 0.5 18.3 ± 0.4*
Creatinine (mg/dL)
Day 4 0.34 ± 0.02 0.30 ± 0.00 0.32 ± 0.01 0.31 ± 0.01 0.32 ± 0.01 0.36 ± 0.02
Day 24 0.30 ± 0.00 0.33 ± 0.02 0.32 ± 0.01 0.32 ± 0.01 0.32 ± 0.01 0.30 ± 0.01
Week 14 0.43 ± 0.02 0.39 ± 0.02 0.41 ± 0.02 0.41 ± 0.01 0.44 ± 0.02 0.43 ± 0.02
Total protein (g/dL)
Day 4 6.0 ± 0.1 6.1 ± 0.1 6.1 ± 0.1 6.0 ± 0.1 6.2 ± 0.1 6.2 ± 0.1
Day 24 6.6 ± 0.1 6.5 ± 0.1 6.5 ± 0.0 6.5 ± 0.1 6.5 ± 0.0 6.3 ± 0.1**
Week 14 7.6 ± 0.1 7.4 ± 0.1 7.5 ± 0.1 7.5 ± 0.1 7.4 ± 0.1 7.4 ± 0.1
Albumin (g/dL)
Day 4 4.1 ± 0.1 4.1 ± 0.1 4.1 ± 0.1 4.1 ± 0.1 4.2 ± 0.1 4.2 ± 0.0
Day 24 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.2 ± 0.0*
Week 14 4.8 ± 0.0 4.7 ± 0.1 4.8 ± 0.1 4.8 ± 0.1 4.8 ± 0.1 4.7 ± 0.1
Globulin (g/dL)
Day 4 1.9 ± 0.0 2.0 ± 0.1 2.0 ± 0.0 1.9 ± 0.0 2.0 ± 0.1** 2.0 ± 0.0**
Day 24 2.3 ± 0.0 2.2 ± 0.1 2.2 ± 0.0 2.2 ± 0.0 2.2 ± 0.0 2.1 ± 0.0*
Week 14 2.9 ± 0.1 2.7 ± 0.1 2.8 ± 0.1 2.7 ± 0.1 2.6 ± 0.1 2.7 ± 0.1
Albumin/globulin ratio
Day 4 2.2 ± 0.0 2.1 ± 0.1* 2.1 ± 0.0** 2.2 ± 0.0* 2.1 ± 0.1** 2.1 ± 0.0**
Day 24 1.9 ± 0.0 2.0 ± 0.0 1.9 ± 0.0 2.0 ± 0.0 2.0 ± 0.0 2.0 ± 0.0
Week 14 1.7 ± 0.0 1.8 ± 0.0 1.7 ± 0.0 1.7 ± 0.0 1.8 ± 0.0 1.8 ± 0.0
Alanine aminotransferase(IU/L)
Day 4 55 ± 1 58 ± 1 59 ± 2 59 ± 2 60 ± 2 60 ± 2
Day 24 48 ± 1 46 ± 1 45 ± 1 47 ± 1 47 ± 1 48 ± 1
Week 14 67 ± 5 62 ± 3 61 ± 2 64 ± 4 63 ± 4 58 ± 2
Alkaline phosphatase (IU/L)
Day 4 698 ± 13 701 ± 17 698 ± 21 705 ± 13 685 ± 15 645 ± 10*
Day 24 511 ± 9 504 ± 14 501 ± 12 488 ± 7 473 ± 9* 468 ± 12**
Week 14 228 ± 3 229 ± 4 227 ± 4 231 ± 5 236 ± 5 228 ± 5
Creatine kinase (IU/L)
Day 4 413 ± 25 584 ± 73 751 ± 119* 715 ± 110* 507 ± 80 638 ± 112
Day 24 307 ± 42 336 ± 69 367 ± 49 312 ± 28 346 ± 43 318 ± 26
Week 14 171 ± 15 192 ± 23 212 ± 30 183 ± 14 268 ± 81 196 ± 25
Sorbitol dehydrogenase (IU/L)
Day 4 23 ± 1 23 ± 1 21 ± 2 22 ± 1 20 ± 2 21 ± 2
Day 24 22 ± 1 19±1 20 ± 1 21 ± 1 19 ± 1 22 ± 1
Week 14 22 ± 1 21 ± 1 22 ± 1 24 ± 2 22 ± 2 23 ± 1
Bile salts (umol/L)
Day 4 26.7 ± 1.4 28.5 ± 2.4 31.4 ± 2.1 26.5±1.7 29.9 ± 1.2 30.3±3.0
Day 24 26.4±2.4 24.6 ± 1.9 24.6 ± 2.6 24.5±1.8 27.7 ± 2.4 25.9 ± 1.7
Week 14 35.1 ± 4.0 26.9 ± 2.9 22.9 ± 3.0 29.6 ± 3.9 31.8±2.7 27.4 ± 2.6
Female
n
Day 4 10 9 10 10 10 10
Day 24 10 10 10 9 10 10
Week 14 10 10 10 10 10 10
Hematology
Hematocrit (%)
Day 4 42.8±0.7 44.4 ± 1.2 43.1 ± 0.7 44.3 ± 1.4 43.4 ± 1.1 42.8±1.0
Day 24 44.9 ± 0.3 45.1 ± 0.4 47.0 ± 0.7* 46.0 ± 0.4 45.6 ± 0.2 45.3±0.4
Week 14 39.0±0.8 38.7 ± 0.5 38.5 ± 0.6 39.2 ± 0.5 39.3±0.4 38.5±0.7
Hemoglobin (g/dL)
Day 4 14.9 ± 0.2 15.4 ± 0.4 14.9 ± 0.2 15.4 ± 0.5 15.0 ± 0.4 14.9 ± 0.3
Day 24 15.9 ± 0.1 16.0 ± 0.1 16.6 ± 0.2* 16.3±0.2 16.0 ± 0.1 16.1 ± 0.2
Week 14 15.1 ± 0.2 15.0 ± 0.2 15.1 ± 0.1 15.4 ± 0.2 15.5±0.2 15.0±0.3
Erythrocytes (106/uL)
Day 4 7.74 ± 0.11 8.02 ± 0.23 7.79 ± 0.10 8.03 ± 0.29 7.82 ± 0.17 7.73±0.17
Day 24 8.28 ± 0.06 8.23±0.08 8.57 ± 0.12 8.40 ± 0.07 8.35 ± 0.05 8.32 ± 0.07
Week 14 7.89 ± 0.12 7.82 ± 0.10 7.88 ± 0.10 7.99 ± 0.12 8.07 ± 0.08 7.87 ± 0.12
Reticulocytes (106/uL)
Day 4 0.59 ± 0.02 0.55 ± 0.04 0.53 ± 0.02 0.58 ± 0.05 0.54 ± 0.03 0.54 ± 0.03
Day 24 0.25 ± 0.01 0.27 ± 0.02 0.22 ± 0.02 0.23 ± 0.02 0.24 ± 0.02 0.22 ± 0.02
Week 14 0.22 ± 0.02 0.21 ± 0.02 0.23 ± 0.01 0.21 ± 0.01 0.21 ± 0.02 0.20 ± 0.02
Mean cell volume (fL)
Day 4 55.2 ± 0.1 55.3 ± 0.2 55.4 ± 0.3 55.6 ± 0.2 55.5±0.2 55.4 ± 0.2
Day 24 54.4 ± 0.2 54.7 ± 0.2 54.9 ± 0.2 54.8 ± 0.2 54.6 ± 0.3 54.4 ± 0.2
Week 14 49.3±0.4 49.5 ± 0.4 48.8±0.3 48.9 ± 0.5 48.7 ± 0.3 49.1 ± 0.4
Mean cell hemoglobin (pg)
Day 4 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.3 ± 0.1 19.2 ± 0.1 19.3 ± 0.1
Day 24 19.2 ± 0.1 19.4 ± 0.1 19.4 ± 0.1 19.4 ± 0.1 19.2 ± 0.1 19.3 ± 0.1
Week 14 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.1 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4 34.8±0.1 34.7 ± 0.1 34.6 ± 0.1 34.8 ± 0.1 34.6 ± 0.0 34.8±0.0
Day 24 35.5 ± 0.1 35.4 ± 0.1 35.3±0.1 35.4 ± 0.1 35.2 ± 0.1* 35.4 ± 0.1
Week 14 38.9 ± 0.4 38.7 ± 0.4 39.3±0.3 39.2 ± 0.4 39.3±0.3 39.0 ± 0.3
Platelets (103/uL)
Day 4 688.0±30.2 637.1 ± 27.6 663.8 ± 23.5 694.5 ± 13.9 689.4 ± 16.8 666.9 ± 22.8
Day 24 609.6 ± 15.7 627.2 ± 18.9 543.9 ± 41.9 575.3 ± 24.4 548.1 ± 16.1 552.8±26.3
Week 14 567.4 ± 20.7 593.8 ± 23.7 614.9 ± 19.3 611.8±24.1 593.2 ± 14.8 562.5 ± 30.4
Leukocytes(103/uL)
Day4 11.99 ± 0.59 11.13±0.59 10.29 ± 0.50 10.27 ± 0.40 11.93 ± 0.77 12.02 ± 0.45
Day24 12.59 ± 0.46 11.59 ± 0.39 11.36 ± 0.64 12.20 ± 0.62 11.87 ± 0.68 12.88 ± 0.49
Week 14 5.02 ± 0.55 4.94 ± 0.37 5.83 ± 0.29 5.91 ± 0.38 6.16 ± 0.39 5.61 ± 0.44
Segmented neutrophils (103/uL)
Day 4 1.17 ± 0.13 0.94 ± 0.07 0.81 ± 0.09 0.72 ± 0.08* 1.21 ± 0.12 1.27 ± 0.14
Day 24 1.01 ± 0.09 1.22 ± 0.11 0.96 ± 0.08 1.14 ± 0.16 1.10 ± 0.10 1.44 ± 0.11*
Week 14 0.50±0.07 0.56±0.03 0.63 ± 0.09 0.85 ± 0.15* 0.81 ± 0.10* 0.82 ± 0.09**
Lymphocytes (103/uL)
Day 4 10.16±0.52 9.59 ± 0.53 8.82 ± 0.45 8.99 ± 0.32 10.05±0.73 10.08 ± 0.42
Day 24 10.60±0.37 9.56±0.36 9.75 ± 0.63 10.33±0.60 10.06 ± 0.63 10.80 ± 0.49
Week 14 4.22 ± 0.45 4.16±0.34 4.96 ± 0.21 4.78 ± 0.31 5.11 ± 0.31 4.53 ± 0.41
Monocytes (103/uL)
Day 4 0.66±0.10 0.59 ± 0.08 0.64 ± 0.10 0.53 ± 0.09 0.63 ± 0.06 0.66 ± 0.09
Day 24 0.88 ± 0.12 0.74 ± 0.08 0.59 ± 0.08 0.67 ± 0.08 0.66 ± 0.08 0.57 ± 0.08
Week 14 0.26±0.04 0.19 ± 0.04 0.19 ± 0.03 0.21 ± 0.04 0.18 ± 0.04 0.23 ± 0.04
Basophils (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 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 0.01 ± 0.01 0.01 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.04 ± 0.02 0.01 ± 0.01
Day 24 0.11 ± 0.04 0.07 ± 0.02 0.06 ± 0.02 0.06 ± 0.03 0.06 ± 0.02 0.07 ± 0.03
Week 14 0.03 ± 0.02 0.02 ± 0.01 0.04 ± 0.02 0.04 ± 0.02 0.06 ± 0.02 0.02 ± 0.01
Clinical Chemistry
Urea nitrogen (mg/dL)
Day 4 13.6±0.7 14.6±1.0 14.3 ± 0.7 14.3±0.6 13.0 ± 0.7 13.4 ± 0.4
Day 24 15.7 ± 0.3 15.6 ± 0.7 15.6 ± 0.7 16.3±0.6 15.8 ± 0.4 15.2 ± 0.4
Week 14 18.8±0.8 19.1 ± 0.7 18.4 ± 0.6 17.6 ± 0.7 19.0 ± 0.6 18.9 ± 1.1
Creatinine (mg/dL)
Day 4 0.34 ± 0.02 0.32 ± 0.01 0.30 ± 0.01 0.34 ± 0.02 0.31 ± 0.02 0.36 ± 0.02
Day 24 0.30±0.00 0.32 ± 0.01 0.35 ± 0.02 0.30 ± 0.00 0.35 ± 0.02 0.31 ± 0.02
Week 14 0.42 ± 0.01 0.45 ± 0.02 0.42 ± 0.01 0.42 ± 0.01 0.44 ± 0.02 0.42 ± 0.02
Total protein (g/dL)
Day 4 6.3 ± 0.1 6.2 ± 0.1 6.3 ± 0.1 6.4 ± 0.1 6.2 ± 0.1 6.3 ± 0.1
Day 24 6.4 ± 0.1 6.4 ± 0.1 6.6 ± 0.1 6.6 ± 0.1 6.7 ± 0.1* 6.4 ± 0.1
Week 14 6.8 ± 0.1 7.1 ± 0.1 7.0 ± 0.1 7.0 ± 0.1 7.1 ± 0.1 6.8 ± 0.1
Albumin (g/dL)
Day 4 4.4 ± 0.0 4.4 ± 0.1 4.5 ± 0.0 4.4 ± 0.1 4.4 ± 0.1 4.4 ± 0.0
Day 24 4.4 ± 0.0 4.5 ± 0.0 4.5 ± 0.1 4.6 ± 0.0 4.6 ± 0.0** 4.5 ± 0.1
Week 14 4.6±0.0 4.8 ± 0.1 4.8 ± 0.1 4.7 ± 0.1 4.7 ± 0.1 4.6 ± 0.1
Globulin (g/dL)
Day 4 1.9 ± 0.1 1.8±0.0 1.9 ± 0.1 2.0 ± 0.1 1.9 ± 0.1 1.9 ± 0.1
Day 24 2.0±0.0 2.0 ± 0.0 2.0 ± 0.0 2.1 ± 0.0 2.0 ± 0.0 2.0 ± 0.0
Week 14 2.2 ± 0.0 2.3 ± 0.0 2.2 ± 0.1 2.3 ± 0.1 2.3 ± 0.1 2.2 ± 0.1
Albumin/globulin ratio
Day 4 2.3 ± 0.1 2.4 ± 0.0 2.4 ± 0.1 2.3 ± 0.1 2.3±0.1 2.4±0.0
Day 24 2.3 ± 0.1 2.3±0.0 2.2 ± 0.0 2.2±0.0 2.3±0.0 2.3±0.1
Week 14 2.1 ± 0.0 2.1 ± 0.0 2.2 ± 0.1 2.1 ± 0.1 2.0±0.0 2.2 ± 0.1
Alanine aminotransferase (IU/L)
Day 4 47 ± 2 48 ± 1 46 ± 1 54 ± 3 47 ± 1 53 ± 2
Day 24 40 ± 1 38 ± 2 41 ± 2 37 ± 1 39 ± 1 40 ± 1
Week 14 61 ± 5 81 ± 10 64 ± 4 57 ± 4 62 ± 3 59 ± 2
Alkaline phosphatase (IU/L)
Day 4 581 ± 17 565 ± 13 561 ± 8 567 ± 8 551 ± 17 551 ± 10
Day 24 390 ± 4 393 ± 10 400 ± 9 389 ± 11 391 ± 7 403 ± 12
Week 14 194 ± 3 188 ± 4 186±5 195 ± 6 186±5 184 ± 5
Creatine kinase (IU/L)
Day 4 736 ± 257 570±139 493 ± 94 1,281 ± 404 452 ± 73 602 ± 84
Day 24 295 ± 30 378 ± 103 231 ± 24 350 ± 56 253±25 448 ± 112
Week 14 251 ± 48 198 ± 25 212 ± 24 226 ± 29 225 ± 37 210 ± 38
Sorbitol dehydrogenase (IU/L)
Day 4 15±2 18 ± 2 15±2 13±2 17 ± 2 13 ± 2
Day 24 22 ± 1 21 ± 1 23 ± 2 20 ± 1 22 ± 1 21 ± 2
Week 14 22 ± 2 28 ± 2* 25 ± 1 24 ± 2 25 ± 2 21 ± 1
Bile salts(umol/L)
Day 4 25.3±2.0 27.7 ± 4.3 23.8±1.1 25.7 ± 2.6 22.5±1.9 23.9 ± 3.1
Day 24 26.4 ± 2.2 23.2 ± 2.3 20.3±1.8 19.9 ± 1.8 20.8 ± 2.1 24.9 ± 2.5
Week 14 34.9 ± 3.2 36.9 ± 2.9 38.5 ± 5.4 32.5 ± 2.6 31.3 ± 3.4 35.8±2.7
*  Significantly different (P<0.05) from the vehicle control group by Dunn's or Shirley's test
** P<0.01
a  Data are presented as mean ± standard error. Statistical tests were performed on unrounded data.

TABLE 5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketonea
  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± 4b 183±4 174 ± 2 175 ± 3 176 ± 2
Heart
Absolute 0.66±0.01 0.66 ± 0.02 0.67 ± 0.01 0.67 ± 0.01 0.65 ± 0.01 0.67 ± 0.01
Relative 3.689 ± 0.063 3.681 ± 0.065 3.644 ± 0.054 3.854 ± 0.068 3.729 ± 0.063 3.778 ± 0.033
R. Kidney
Absolute 0.73±0.03 0.70 ± 0.02 0.75 ± 0.02 0.75 ± 0.02 0.72 ± 0.02 0.75 ± 0.02
Relative 4.043±0.133 3.879 ± 0.052 4.074 ± 0.078 4.309 ± 0.131 4.091 ± 0.093 4.292 ± 0.116
Liver
Absolute 6.11±0.20 6.25 ± 0.18 6.37 ± 0.16 6.11 ± 0.11 6.16±0.15 6.35 ± 0.10
Relative 33.963 ± 0.728 34.688 ± 0.352 34.705±0.442 35.220 ± 0.437 35.102±0.525 36.106±0.393**
Lung
Absolute 1.10±0.04 1.08 ± 0.05 1.09±0.03 1.06±0.05 1.06±0.03 1.06±0.03
Relative 6.107±0.213 5.991 ± 0.233 5.961 ± 0.111 6.114±0.241 6.062 ± 0.171 6.030 ± 0.138
Thymus
Absolute 0.225 ± 0.012 0.226 ± 0.010 0.224 ± 0.007 0.206 ± 0.006 0.225 ± 0.011 0.215±0.007
Relative 1.253 ± 0.069 1.255 ± 0.033 1.221 ± 0.040 1.187±0.036 1.285 ± 0.063 1.223 ± 0.032
*  Significantly different (P<0.05) from the vehicle control group by Williams' test
** P<0.01
a  Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).
bn=10

Table 6 Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketonea
  Vehicle Control 87.5 mg/kg 175 mg/kg 350 mg/kg
n 10 10 10 10
Weights (g)
Necropsy body wt 309 ± 6 300 ± 8 284±6** 278 ± 7**
L. Cauda epididymis 0.1669±0.0105 0.1537±0.0037 0.1471 ± 0.0042 0.1446±0.0064
L. Epididymis 0.4256 ± 0.0054 0.4358 ± 0.0076 0.4262 ± 0.0100 0.4017±0.0076
L. Testis 1.4617±0.0287 1.4516±0.0237 1.4483 ± 0.0242 1.3924 ± 0.0290
Spermatid measurements
Spermatid heads (106/testis) 171.38 ± 7.95 169.00 ± 6.25 174.25 ± 8.45 155.38±8.39
Spermatid heads (103/mg testis) 123.0 ± 4.5 123.3 ± 4.7 126.9 ± 4.7 118.4±5.5
Epididymalspermatozoal measurements
Sperm motility (%) 84.1 ± 0.8 85.3 ± 1.4 83.9 ± 0.8 83.8 ± 0.7
Sperm (106/cauda epididymis) 55.4 ± 10.4 46.3 ± 9.4 47.4 ± 8.3 36.6 ± 5.5
Sperm (103/mg cauda epididymis) 333 ± 62 301 ± 63 333 ± 63 255 ± 38
** Significantly different (P<0.01) from the vehicle control group by Williams' test  
a  Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by DUnnett's test (tissue weights) or Dunn's test (spermatid and epididymal spermatozoal measurements).
Table 7 Estrous Cycle Characterization for Female Rats in the 3-Month Dermal Study of Methyl trans-Styryl Ketonea
  Vehicle Control 87.5 mg/kg 175 mg/kg 350 mg/kg
Number weighed at necropsy 10 10 10 10
Necropsy body wt (g) 180 ± 4 174 ± 2 175 ± 3 176 ± 2
Proportion of regular cycling femalesb 10/10 9/10 10/10 10/10
Estrous cycle length (days) 5.0 ± 0.05 4.9±0.08 5.0 ± 0.00 4.9 ± 0.07
Estrous stages (% of cycle)
Diestrus 59.2 57.5 60.0 62.5
Proestrus 17.5 15.0 14.2 15.8
Estrus 20.8 25.8 25.8 20.0
Metestrus 2.5 1.7 0.0 1.7
a  Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnett's test (body weight) or Dunn's test (estrous cycle length). By multivariate analysis of variance, dosed females do not differ significantly from the vehicle control females in the relative length of time spent in the estrous stages. The tests for equality of transition probability matrices among all groups and between the vehicle control group and each dosed group indicated there were no significant differences.
b  Number of females with a regular cycle/number of females cycling
Conclusions:
The study was performed with the related 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. An NOEAL was derived (87.5 mg/kg bw).
Executive summary:

The related 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:
No repeated dose toxicity studies are available for benzalacetone. Therefore, a subchronic study in rats conducted with the related substance methyl-trans-styryl ketone, the nearest analogue, 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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This information comes from an NTP-report (TR 572), which deals with the related substance: methyl trans-styryl ketone. The quality of this report is considered to be high.
Qualifier:
equivalent or similar to
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
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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
Remarks:
Doses / Concentrations:
22 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
44 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
87.5 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
175 mg/ kg bw/ day
Basis:
nominal per unit body weight
Remarks:
Doses / Concentrations:
350 mg/ kg bw/ day
Basis:
nominal per unit body weight
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.
Dose descriptor:
NOAEL
Effect level:
87.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
TABLE 2
Survival and Body Weights of Rats in the 3-Month Dermal Study of Methyl trans-Styryl Ketonea
Dose (mg/kg) Survivalb initial Body Weight (g) Final Body Weight (g) Change in Body Weight (g) Final Weight Relative to Controls(%)
Male          
0 10/10 100±2 309 ± 6 209 ± 5
22 10/10 100±3 311 ± 7 211 ± 6 100
44 10/10 100 ± 3 308 ± 8 208 ± 6 99
87.5 10/10 101 ± 3 300 ± 8 200 ± 5 97
175 10/10 99±3 284±6* 185 ± 4** 92
350 10/10 99±3 278±7** 179 ± 5** 90
Female
0 10/10 94 ± 1 180 ± 4 86 ± 3
22 10/10 93 ± 2 180 ± 4 87 ± 3 100
44 10/10 93 ± 2 183 ± 4 90 ± 3 102
87.5 10/10 94 ± 2 174 ± 2 80 ± 2 97
175 10/10 93 ± 2 175 ± 3 83 ± 3 98
350 10/10 94 ± 1 176 ± 2 82 ± 3 98
*  Significantly different (P<0.05) from the vehicle control group by William's test
** P<0.01
a  Weights and weight changes are given as mean ± standard error.
b  Number of animals surviving at 14 weeks/number initially in group

TABLE 3
Incidences of Selected Nonneoplastic Lesions in Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketone
  Vehicle Control 22 mg/kg 44 mg/kg 87.5 mg/kg 175 mg/kg 350 mg/kg
Male
Nosea 10 10 10 10 10 10
Respiratory Epithelium,
Hyperplasia, Goblet Cellb 2 (1.0)c 6 (1.3) 1 (1.0) 6 (1.2) 4 (1.3) 9** (1.7)
Skin, Site of Application 10 10 9 10 10 10
Sebaceous Gland,
Hypertrophy 0 1 (1.0) 3 (1.0) 6** (1.2) 10** (1.8) 7** (1.9)
Epidermis, Hyperplasia 0 3 (1.0) 6** (1.0) 8** (1.5) 10** (2.2) 10** (2.3)
Hyperkeratosis 1 (1.0) 6* (1.2) 7** (1.4) 10** (1.7) 10** (1.9) 9** (2.1)
Inflammation, Chronic Active 0 0 0 0 3 (1.7) 4*(2.5)
Necrosis 0 0 0 0 3 (2.0) 3 (2.3)
Female
Nose 10 10 10 10 10 10
Respiratory Epithelium,
Hyperplasia, Goblet Cell 0 8** (1.0) 3 (1.0) 3 (1.0) 7** (1.4) 5*(1.2)
Skin, Site of Application 10 10 10 10 10 10
Sebaceous Gland, Hypertrophy                         0                     1   (1.0)          5*  (1.0)          7** (1.0)          4*  (1.5)          8** (1.6)
Epidermis, Degeneration 0 0 0 0 2 (3.0) 1 (1.0)
Epidermis, Hyperplasia 0 3 (1.0) 3 (1.0) 7** (1.6) 9** (2.2) 10** (2.2)
Hyperkeratosis 0 6** (1.0) 6** (1.0) 9** (1.9) 8** (1.6) 9** (2.0)
Inflammation, Chronic Active 0 0 0 1 (1.0) 6**(2.5) 4* (2.8)
Necrosis 0 0 0 0 4* (2.0)
Ulcer 0 0 0 0 1 1
*  Significantly different (P<0.05) from the vehicle control group by the Fisher exact test
** P<0.01
a  Number of animals with tissue examined microscopically
b  Number of animals with lesion
c  Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

TABLE 4 Haematology and Clinical Chemistry Data for Rats in the 3-Month Dermal Study of Methyl trans-Styryl Ketonea
  Vehicle Control 22 mg/kg 44 mg/kg 87.5 mg/kg 175 mg/kg 350 mg/kg
Male
Haematology
n Day 4 10 10 10 9 10 10
Day 24 10 10 10 10 10 10
Week14 10 10 10 10 10 10
Haematocrit(%)
Day 4 38.5 ± 0.5 37.5 ± 1.0 38.3±0.3 38.4 ± 0.5 38.7 ± 0.2 38.6 ± 0.4
Day 24 43.8±0.5 43.9 ± 0.7 43.9 ± 0.4 43.7 ± 0.5 43.0 ± 0.3 43.4 ± 0.5
Week14 44.8 ± 0.4 44.1 ± 0.5 44.8 ± 0.4 44.6 ± 0.4 44.6 ± 0.7 44.8 ± 0.5
Haemoglobin (g/dL)
Day 4 13.2 ± 0.2 12.9 ± 0.3 13.2 ± 0.1 13.2 ± 0.1 13.3 ± 0.1 13.2 ± 0.1
Day 24 15.4 ± 0.2 15.5±0.2 15.4 ± 0.1 15.3±0.1 15.0 ± 0.1 15.2 ± 0.2
Week 14 15.6±0.1 15.4 ± 0.1 15.7 ± 0.1 15.6 ± 0.1 15.5±0.1 15.7 ± 0.1
Erythrocytes (106/uL)
Day 4 6.93 ± 0.08 6.78 ± 0.16 6.90 ± 0.07 6.92 ± 0.09 7.04 ± 0.04 7.07 ± 0.07
Day 24 8.02 ± 0.10 8.02 ± 0.11 8.03 ± 0.08 8.00 ± 0.10 7.88 ± 0.05 7.89 ± 0.09
Week 13 8.93 ± 0.06 8.87 ± 0.08 8.99 ± 0.04 8.93 ± 0.07 8.87 ± 0.09 8.98 ± 0.08
Reticulocytes (106/uL)
Day 4 0.59 ± 0.03 0.58 ± 0.03 0.60 ± 0.02 0.58 ± 0.01 0.56 ± 0.02 0.63 ± 0.01
Day 24 0.40±0.04 0.41 ± 0.03 0.41 ± 0.02 0.32 ± 0.01 0.37 ± 0.03 0.38 ± 0.03
Week 14 0.24 ± 0.02 0.19 ± 0.02 0.21 ± 0.02 0.25 ± 0.01 0.22 ± 0.02 0.25 ± 0.01
Mean cell volume (fL)
Day 4 55.4 ± 0.2 55.3 ± 0.3 55.5 ± 0.2 55.2 ± 0.2 54.8 ± 0.3 54.6 ± 0.3
Day 24 54.6±0.2 54.7 ± 0.2 54.7 ± 0.2 54.6 ± 0.2 54.7 ± 0.2 55.0 ± 0.2
Week 14 50.1 ± 0.2 49.7 ± 0.3 49.7 ± 0.4 49.9 ± 0.4 50.3 ± 0.3 49.8 ± 0.1
Mean cell hemoglobin (pg)
Day 4 19.0 ± 0.1 19.1 ± 0.1 19.2 ± 0.1 19.1 ± 0.1 18.9 ± 0.1 18.7 ± 0.1*
Day 24 19.1 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.0 ± 0.0 19.2 ± 0.1
Week 14 17.4 ± 0.1 17.4 ± 0.0 17.5 ± 0.0 17.5 ± 0.1 17.5 ± 0.1 17.5 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4 34.2 ± 0.1 34.4 ± 0.1 34.6 ± 0.1 34.5 ± 0.1 34.4 ± 0.1 34.2 ± 0.1
Day 24 35.0±0.1 35.2 ± 0.1 35.0 ± 0.1 35.0 ± 0.1 34.8 ± 0.1 34.9 ± 0.1
Week 14 34.8±0.2 34.9 ± 0.2 35.1 ± 0.2 35.0 ± 0.2 34.8 ± 0.2 35.0 ± 0.2
Platelets (103/uL)
Day 4 727.3 ± 14.5 706.1 ± 19.1 685.8 ± 35.6 745.6 ± 18.4 743.8 ± 9.6 742.7 ± 19.2
Day 24 563.8 ± 20.0 546.3 ± 23.8 536.1 ± 27.9 552.3 ± 21.6 569.5 ± 11.3 518.0 ± 27.7
Week 14 601.9 ± 10.9 514.0±19.5** 541.2 ± 19.9 535.5 ± 14.4* 546.6 ± 21.6 563.5 ± 16.9
Leukocytes (103/uL)
Day 4 8.72 ± 0.42 8.29 ± 0.56 9.43 ± 0.72 9.67 ± 0.59 10.54 ± 0.77 9.69 ± 0.64
Day 24 12.17 ± 0.54 11.71 ± 0.46 12.48 ± 0.35 12.26 ± 0.41 12.26 ± 0.29 12.06 ± 0.54
Week 14 7.80±0.44 7.62 ± 0.38 8.46 ± 0.31 8.90 ± 0.37 8.28 ± 0.32 7.94 ± 0.44
Segmented neutrophils (103/uL)
Day 4 0.79 ± 0.12 0.89 ± 0.08 0.94 ± 0.14 1.02 ± 0.12 1.48 ± 0.18** 1.95 ± 0.16**
Day 24 1.09 ± 0.14 0.88 ± 0.08 1.30 ± 0.14 1.13±0.17 1.27 ± 0.11 1.24 ± 0.12
Week 14 1.02 ± 0.13 0.98 ± 0.12 1.13±0.10 1.17 ± 0.09 1.16 ± 0.09 1.23 ± 0.12
Lymphocytes (103/uL)
Day 4 7.40±0.33 6.88 ± 0.49 7.89 ± 0.58 8.04 ± 0.51 8.33 ± 0.57 7.19 ± 0.50
Day 24 10.42 ± 0.46 10.05 ± 0.40 10.40 ± 0.33 10.45 ± 0.30 10.10 ± 0.30 10.12 ± 0.43
Week 14 6.48 ± 0.40 6.25 ± 0.32 6.89 ± 0.26 7.17 ± 0.32 6.71 ± 0.35 6.35 ± 0.38
Monocytes (103/uL)
Day 4 0.50±0.06 0.48 ± 0.08 0.58 ± 0.05 0.56 ± 0.08 0.71 ± 0.09 0.50 ± 0.07
Day 24 0.63 ± 0.11 0.72 ± 0.10 0.77 ± 0.09 0.65 ± 0.10 0.83 ± 0.06 0.64 ± 0.11
Week 14 0.22 ± 0.02 0.36 ± 0.07 0.34 ± 0.06 0.38 ± 0.08 0.30 ± 0.04 0.31 ± 0.06
Basophils(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 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 0.04 ± 0.02 0.40 ± 0.02 0.03 ± 0.02 0.05 ± 0.03 0.02 ± 0.01 0.04 ± 0.02
Day 24 0.04 ± 0.02 0.07 ± 0.02 0.01 ± 0.01 0.04 ± 0.02 0.06 ± 0.03 0.06 ± 0.03
Week 14 0.05 ± 0.02 0.02 ± 0.01 0.08 ± 0.03 0.12 ± 0.02 0.06 ± 0.03 0.04 ± 0.01
Clinical Chemistry
n 10 10 10 10 10 10
Ureanitrogen(mg/dL)
Day 4 14.4 ± 0.6 14.6 ± 0.3 13.6 ± 0.6 14.6 ± 0.5 13.9 ± 0.4 14.9 ± 0.5
Day 24 13.1 ± 0.4 12.8 ± 0.4 12.5 ± 0.4 11.7 ± 0.6 12.6 ± 0.5 12.1 ± 0.4
Week 14 16.4 ± 0.5 16.8 ± 0.6 16.7 ± 0.6 17.2 ± 1.1 17.5 ± 0.5 18.3 ± 0.4*
Creatinine (mg/dL)
Day 4 0.34 ± 0.02 0.30 ± 0.00 0.32 ± 0.01 0.31 ± 0.01 0.32 ± 0.01 0.36 ± 0.02
Day 24 0.30 ± 0.00 0.33 ± 0.02 0.32 ± 0.01 0.32 ± 0.01 0.32 ± 0.01 0.30 ± 0.01
Week 14 0.43 ± 0.02 0.39 ± 0.02 0.41 ± 0.02 0.41 ± 0.01 0.44 ± 0.02 0.43 ± 0.02
Total protein (g/dL)
Day 4 6.0 ± 0.1 6.1 ± 0.1 6.1 ± 0.1 6.0 ± 0.1 6.2 ± 0.1 6.2 ± 0.1
Day 24 6.6 ± 0.1 6.5 ± 0.1 6.5 ± 0.0 6.5 ± 0.1 6.5 ± 0.0 6.3 ± 0.1**
Week 14 7.6 ± 0.1 7.4 ± 0.1 7.5 ± 0.1 7.5 ± 0.1 7.4 ± 0.1 7.4 ± 0.1
Albumin (g/dL)
Day 4 4.1 ± 0.1 4.1 ± 0.1 4.1 ± 0.1 4.1 ± 0.1 4.2 ± 0.1 4.2 ± 0.0
Day 24 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.3 ± 0.0 4.2 ± 0.0*
Week 14 4.8 ± 0.0 4.7 ± 0.1 4.8 ± 0.1 4.8 ± 0.1 4.8 ± 0.1 4.7 ± 0.1
Globulin (g/dL)
Day 4 1.9 ± 0.0 2.0 ± 0.1 2.0 ± 0.0 1.9 ± 0.0 2.0 ± 0.1** 2.0 ± 0.0**
Day 24 2.3 ± 0.0 2.2 ± 0.1 2.2 ± 0.0 2.2 ± 0.0 2.2 ± 0.0 2.1 ± 0.0*
Week 14 2.9 ± 0.1 2.7 ± 0.1 2.8 ± 0.1 2.7 ± 0.1 2.6 ± 0.1 2.7 ± 0.1
Albumin/globulin ratio
Day 4 2.2 ± 0.0 2.1 ± 0.1* 2.1 ± 0.0** 2.2 ± 0.0* 2.1 ± 0.1** 2.1 ± 0.0**
Day 24 1.9 ± 0.0 2.0 ± 0.0 1.9 ± 0.0 2.0 ± 0.0 2.0 ± 0.0 2.0 ± 0.0
Week 14 1.7 ± 0.0 1.8 ± 0.0 1.7 ± 0.0 1.7 ± 0.0 1.8 ± 0.0 1.8 ± 0.0
Alanine aminotransferase(IU/L)
Day 4 55 ± 1 58 ± 1 59 ± 2 59 ± 2 60 ± 2 60 ± 2
Day 24 48 ± 1 46 ± 1 45 ± 1 47 ± 1 47 ± 1 48 ± 1
Week 14 67 ± 5 62 ± 3 61 ± 2 64 ± 4 63 ± 4 58 ± 2
Alkaline phosphatase (IU/L)
Day 4 698 ± 13 701 ± 17 698 ± 21 705 ± 13 685 ± 15 645 ± 10*
Day 24 511 ± 9 504 ± 14 501 ± 12 488 ± 7 473 ± 9* 468 ± 12**
Week 14 228 ± 3 229 ± 4 227 ± 4 231 ± 5 236 ± 5 228 ± 5
Creatine kinase (IU/L)
Day 4 413 ± 25 584 ± 73 751 ± 119* 715 ± 110* 507 ± 80 638 ± 112
Day 24 307 ± 42 336 ± 69 367 ± 49 312 ± 28 346 ± 43 318 ± 26
Week 14 171 ± 15 192 ± 23 212 ± 30 183 ± 14 268 ± 81 196 ± 25
Sorbitol dehydrogenase (IU/L)
Day 4 23 ± 1 23 ± 1 21 ± 2 22 ± 1 20 ± 2 21 ± 2
Day 24 22 ± 1 19±1 20 ± 1 21 ± 1 19 ± 1 22 ± 1
Week 14 22 ± 1 21 ± 1 22 ± 1 24 ± 2 22 ± 2 23 ± 1
Bile salts (umol/L)
Day 4 26.7 ± 1.4 28.5 ± 2.4 31.4 ± 2.1 26.5±1.7 29.9 ± 1.2 30.3±3.0
Day 24 26.4±2.4 24.6 ± 1.9 24.6 ± 2.6 24.5±1.8 27.7 ± 2.4 25.9 ± 1.7
Week 14 35.1 ± 4.0 26.9 ± 2.9 22.9 ± 3.0 29.6 ± 3.9 31.8±2.7 27.4 ± 2.6
Female
n
Day 4 10 9 10 10 10 10
Day 24 10 10 10 9 10 10
Week 14 10 10 10 10 10 10
Hematology
Hematocrit (%)
Day 4 42.8±0.7 44.4 ± 1.2 43.1 ± 0.7 44.3 ± 1.4 43.4 ± 1.1 42.8±1.0
Day 24 44.9 ± 0.3 45.1 ± 0.4 47.0 ± 0.7* 46.0 ± 0.4 45.6 ± 0.2 45.3±0.4
Week 14 39.0±0.8 38.7 ± 0.5 38.5 ± 0.6 39.2 ± 0.5 39.3±0.4 38.5±0.7
Hemoglobin (g/dL)
Day 4 14.9 ± 0.2 15.4 ± 0.4 14.9 ± 0.2 15.4 ± 0.5 15.0 ± 0.4 14.9 ± 0.3
Day 24 15.9 ± 0.1 16.0 ± 0.1 16.6 ± 0.2* 16.3±0.2 16.0 ± 0.1 16.1 ± 0.2
Week 14 15.1 ± 0.2 15.0 ± 0.2 15.1 ± 0.1 15.4 ± 0.2 15.5±0.2 15.0±0.3
Erythrocytes (106/uL)
Day 4 7.74 ± 0.11 8.02 ± 0.23 7.79 ± 0.10 8.03 ± 0.29 7.82 ± 0.17 7.73±0.17
Day 24 8.28 ± 0.06 8.23±0.08 8.57 ± 0.12 8.40 ± 0.07 8.35 ± 0.05 8.32 ± 0.07
Week 14 7.89 ± 0.12 7.82 ± 0.10 7.88 ± 0.10 7.99 ± 0.12 8.07 ± 0.08 7.87 ± 0.12
Reticulocytes (106/uL)
Day 4 0.59 ± 0.02 0.55 ± 0.04 0.53 ± 0.02 0.58 ± 0.05 0.54 ± 0.03 0.54 ± 0.03
Day 24 0.25 ± 0.01 0.27 ± 0.02 0.22 ± 0.02 0.23 ± 0.02 0.24 ± 0.02 0.22 ± 0.02
Week 14 0.22 ± 0.02 0.21 ± 0.02 0.23 ± 0.01 0.21 ± 0.01 0.21 ± 0.02 0.20 ± 0.02
Mean cell volume (fL)
Day 4 55.2 ± 0.1 55.3 ± 0.2 55.4 ± 0.3 55.6 ± 0.2 55.5±0.2 55.4 ± 0.2
Day 24 54.4 ± 0.2 54.7 ± 0.2 54.9 ± 0.2 54.8 ± 0.2 54.6 ± 0.3 54.4 ± 0.2
Week 14 49.3±0.4 49.5 ± 0.4 48.8±0.3 48.9 ± 0.5 48.7 ± 0.3 49.1 ± 0.4
Mean cell hemoglobin (pg)
Day 4 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.3 ± 0.1 19.2 ± 0.1 19.3 ± 0.1
Day 24 19.2 ± 0.1 19.4 ± 0.1 19.4 ± 0.1 19.4 ± 0.1 19.2 ± 0.1 19.3 ± 0.1
Week 14 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.2 ± 0.1 19.1 ± 0.1
Mean cell hemoglobin concentration (g/dL)
Day 4 34.8±0.1 34.7 ± 0.1 34.6 ± 0.1 34.8 ± 0.1 34.6 ± 0.0 34.8±0.0
Day 24 35.5 ± 0.1 35.4 ± 0.1 35.3±0.1 35.4 ± 0.1 35.2 ± 0.1* 35.4 ± 0.1
Week 14 38.9 ± 0.4 38.7 ± 0.4 39.3±0.3 39.2 ± 0.4 39.3±0.3 39.0 ± 0.3
Platelets (103/uL)
Day 4 688.0±30.2 637.1 ± 27.6 663.8 ± 23.5 694.5 ± 13.9 689.4 ± 16.8 666.9 ± 22.8
Day 24 609.6 ± 15.7 627.2 ± 18.9 543.9 ± 41.9 575.3 ± 24.4 548.1 ± 16.1 552.8±26.3
Week 14 567.4 ± 20.7 593.8 ± 23.7 614.9 ± 19.3 611.8±24.1 593.2 ± 14.8 562.5 ± 30.4
Leukocytes(103/uL)
Day4 11.99 ± 0.59 11.13±0.59 10.29 ± 0.50 10.27 ± 0.40 11.93 ± 0.77 12.02 ± 0.45
Day24 12.59 ± 0.46 11.59 ± 0.39 11.36 ± 0.64 12.20 ± 0.62 11.87 ± 0.68 12.88 ± 0.49
Week 14 5.02 ± 0.55 4.94 ± 0.37 5.83 ± 0.29 5.91 ± 0.38 6.16 ± 0.39 5.61 ± 0.44
Segmented neutrophils (103/uL)
Day 4 1.17 ± 0.13 0.94 ± 0.07 0.81 ± 0.09 0.72 ± 0.08* 1.21 ± 0.12 1.27 ± 0.14
Day 24 1.01 ± 0.09 1.22 ± 0.11 0.96 ± 0.08 1.14 ± 0.16 1.10 ± 0.10 1.44 ± 0.11*
Week 14 0.50±0.07 0.56±0.03 0.63 ± 0.09 0.85 ± 0.15* 0.81 ± 0.10* 0.82 ± 0.09**
Lymphocytes (103/uL)
Day 4 10.16±0.52 9.59 ± 0.53 8.82 ± 0.45 8.99 ± 0.32 10.05±0.73 10.08 ± 0.42
Day 24 10.60±0.37 9.56±0.36 9.75 ± 0.63 10.33±0.60 10.06 ± 0.63 10.80 ± 0.49
Week 14 4.22 ± 0.45 4.16±0.34 4.96 ± 0.21 4.78 ± 0.31 5.11 ± 0.31 4.53 ± 0.41
Monocytes (103/uL)
Day 4 0.66±0.10 0.59 ± 0.08 0.64 ± 0.10 0.53 ± 0.09 0.63 ± 0.06 0.66 ± 0.09
Day 24 0.88 ± 0.12 0.74 ± 0.08 0.59 ± 0.08 0.67 ± 0.08 0.66 ± 0.08 0.57 ± 0.08
Week 14 0.26±0.04 0.19 ± 0.04 0.19 ± 0.03 0.21 ± 0.04 0.18 ± 0.04 0.23 ± 0.04
Basophils (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 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 0.01 ± 0.01 0.01 ± 0.01 0.02 ± 0.01 0.02 ± 0.01 0.04 ± 0.02 0.01 ± 0.01
Day 24 0.11 ± 0.04 0.07 ± 0.02 0.06 ± 0.02 0.06 ± 0.03 0.06 ± 0.02 0.07 ± 0.03
Week 14 0.03 ± 0.02 0.02 ± 0.01 0.04 ± 0.02 0.04 ± 0.02 0.06 ± 0.02 0.02 ± 0.01
Clinical Chemistry
Urea nitrogen (mg/dL)
Day 4 13.6±0.7 14.6±1.0 14.3 ± 0.7 14.3±0.6 13.0 ± 0.7 13.4 ± 0.4
Day 24 15.7 ± 0.3 15.6 ± 0.7 15.6 ± 0.7 16.3±0.6 15.8 ± 0.4 15.2 ± 0.4
Week 14 18.8±0.8 19.1 ± 0.7 18.4 ± 0.6 17.6 ± 0.7 19.0 ± 0.6 18.9 ± 1.1
Creatinine (mg/dL)
Day 4 0.34 ± 0.02 0.32 ± 0.01 0.30 ± 0.01 0.34 ± 0.02 0.31 ± 0.02 0.36 ± 0.02
Day 24 0.30±0.00 0.32 ± 0.01 0.35 ± 0.02 0.30 ± 0.00 0.35 ± 0.02 0.31 ± 0.02
Week 14 0.42 ± 0.01 0.45 ± 0.02 0.42 ± 0.01 0.42 ± 0.01 0.44 ± 0.02 0.42 ± 0.02
Total protein (g/dL)
Day 4 6.3 ± 0.1 6.2 ± 0.1 6.3 ± 0.1 6.4 ± 0.1 6.2 ± 0.1 6.3 ± 0.1
Day 24 6.4 ± 0.1 6.4 ± 0.1 6.6 ± 0.1 6.6 ± 0.1 6.7 ± 0.1* 6.4 ± 0.1
Week 14 6.8 ± 0.1 7.1 ± 0.1 7.0 ± 0.1 7.0 ± 0.1 7.1 ± 0.1 6.8 ± 0.1
Albumin (g/dL)
Day 4 4.4 ± 0.0 4.4 ± 0.1 4.5 ± 0.0 4.4 ± 0.1 4.4 ± 0.1 4.4 ± 0.0
Day 24 4.4 ± 0.0 4.5 ± 0.0 4.5 ± 0.1 4.6 ± 0.0 4.6 ± 0.0** 4.5 ± 0.1
Week 14 4.6±0.0 4.8 ± 0.1 4.8 ± 0.1 4.7 ± 0.1 4.7 ± 0.1 4.6 ± 0.1
Globulin (g/dL)
Day 4 1.9 ± 0.1 1.8±0.0 1.9 ± 0.1 2.0 ± 0.1 1.9 ± 0.1 1.9 ± 0.1
Day 24 2.0±0.0 2.0 ± 0.0 2.0 ± 0.0 2.1 ± 0.0 2.0 ± 0.0 2.0 ± 0.0
Week 14 2.2 ± 0.0 2.3 ± 0.0 2.2 ± 0.1 2.3 ± 0.1 2.3 ± 0.1 2.2 ± 0.1
Albumin/globulin ratio
Day 4 2.3 ± 0.1 2.4 ± 0.0 2.4 ± 0.1 2.3 ± 0.1 2.3±0.1 2.4±0.0
Day 24 2.3 ± 0.1 2.3±0.0 2.2 ± 0.0 2.2±0.0 2.3±0.0 2.3±0.1
Week 14 2.1 ± 0.0 2.1 ± 0.0 2.2 ± 0.1 2.1 ± 0.1 2.0±0.0 2.2 ± 0.1
Alanine aminotransferase (IU/L)
Day 4 47 ± 2 48 ± 1 46 ± 1 54 ± 3 47 ± 1 53 ± 2
Day 24 40 ± 1 38 ± 2 41 ± 2 37 ± 1 39 ± 1 40 ± 1
Week 14 61 ± 5 81 ± 10 64 ± 4 57 ± 4 62 ± 3 59 ± 2
Alkaline phosphatase (IU/L)
Day 4 581 ± 17 565 ± 13 561 ± 8 567 ± 8 551 ± 17 551 ± 10
Day 24 390 ± 4 393 ± 10 400 ± 9 389 ± 11 391 ± 7 403 ± 12
Week 14 194 ± 3 188 ± 4 186±5 195 ± 6 186±5 184 ± 5
Creatine kinase (IU/L)
Day 4 736 ± 257 570±139 493 ± 94 1,281 ± 404 452 ± 73 602 ± 84
Day 24 295 ± 30 378 ± 103 231 ± 24 350 ± 56 253±25 448 ± 112
Week 14 251 ± 48 198 ± 25 212 ± 24 226 ± 29 225 ± 37 210 ± 38
Sorbitol dehydrogenase (IU/L)
Day 4 15±2 18 ± 2 15±2 13±2 17 ± 2 13 ± 2
Day 24 22 ± 1 21 ± 1 23 ± 2 20 ± 1 22 ± 1 21 ± 2
Week 14 22 ± 2 28 ± 2* 25 ± 1 24 ± 2 25 ± 2 21 ± 1
Bile salts(umol/L)
Day 4 25.3±2.0 27.7 ± 4.3 23.8±1.1 25.7 ± 2.6 22.5±1.9 23.9 ± 3.1
Day 24 26.4 ± 2.2 23.2 ± 2.3 20.3±1.8 19.9 ± 1.8 20.8 ± 2.1 24.9 ± 2.5
Week 14 34.9 ± 3.2 36.9 ± 2.9 38.5 ± 5.4 32.5 ± 2.6 31.3 ± 3.4 35.8±2.7
*  Significantly different (P<0.05) from the vehicle control group by Dunn's or Shirley's test
** P<0.01
a  Data are presented as mean ± standard error. Statistical tests were performed on unrounded data.

TABLE 5 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketonea
  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± 4b 183±4 174 ± 2 175 ± 3 176 ± 2
Heart
Absolute 0.66±0.01 0.66 ± 0.02 0.67 ± 0.01 0.67 ± 0.01 0.65 ± 0.01 0.67 ± 0.01
Relative 3.689 ± 0.063 3.681 ± 0.065 3.644 ± 0.054 3.854 ± 0.068 3.729 ± 0.063 3.778 ± 0.033
R. Kidney
Absolute 0.73±0.03 0.70 ± 0.02 0.75 ± 0.02 0.75 ± 0.02 0.72 ± 0.02 0.75 ± 0.02
Relative 4.043±0.133 3.879 ± 0.052 4.074 ± 0.078 4.309 ± 0.131 4.091 ± 0.093 4.292 ± 0.116
Liver
Absolute 6.11±0.20 6.25 ± 0.18 6.37 ± 0.16 6.11 ± 0.11 6.16±0.15 6.35 ± 0.10
Relative 33.963 ± 0.728 34.688 ± 0.352 34.705±0.442 35.220 ± 0.437 35.102±0.525 36.106±0.393**
Lung
Absolute 1.10±0.04 1.08 ± 0.05 1.09±0.03 1.06±0.05 1.06±0.03 1.06±0.03
Relative 6.107±0.213 5.991 ± 0.233 5.961 ± 0.111 6.114±0.241 6.062 ± 0.171 6.030 ± 0.138
Thymus
Absolute 0.225 ± 0.012 0.226 ± 0.010 0.224 ± 0.007 0.206 ± 0.006 0.225 ± 0.011 0.215±0.007
Relative 1.253 ± 0.069 1.255 ± 0.033 1.221 ± 0.040 1.187±0.036 1.285 ± 0.063 1.223 ± 0.032
*  Significantly different (P<0.05) from the vehicle control group by Williams' test
** P<0.01
a  Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ weight/g body weight (mean ± standard error).
bn=10

Table 6 Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Dermal Study of Methyltrans-Styryl Ketonea
  Vehicle Control 87.5 mg/kg 175 mg/kg 350 mg/kg
n 10 10 10 10
Weights (g)
Necropsy body wt 309 ± 6 300 ± 8 284±6** 278 ± 7**
L. Cauda epididymis 0.1669±0.0105 0.1537±0.0037 0.1471 ± 0.0042 0.1446±0.0064
L. Epididymis 0.4256 ± 0.0054 0.4358 ± 0.0076 0.4262 ± 0.0100 0.4017±0.0076
L. Testis 1.4617±0.0287 1.4516±0.0237 1.4483 ± 0.0242 1.3924 ± 0.0290
Spermatid measurements
Spermatid heads (106/testis) 171.38 ± 7.95 169.00 ± 6.25 174.25 ± 8.45 155.38±8.39
Spermatid heads (103/mg testis) 123.0 ± 4.5 123.3 ± 4.7 126.9 ± 4.7 118.4±5.5
Epididymalspermatozoal measurements
Sperm motility (%) 84.1 ± 0.8 85.3 ± 1.4 83.9 ± 0.8 83.8 ± 0.7
Sperm (106/cauda epididymis) 55.4 ± 10.4 46.3 ± 9.4 47.4 ± 8.3 36.6 ± 5.5
Sperm (103/mg cauda epididymis) 333 ± 62 301 ± 63 333 ± 63 255 ± 38
** Significantly different (P<0.01) from the vehicle control group by Williams' test  
a  Data are presented as mean ± standard error. Differences from the vehicle control group are not significant by DUnnett's test (tissue weights) or Dunn's test (spermatid and epididymal spermatozoal measurements).
Table 7 Estrous Cycle Characterization for Female Rats in the 3-Month Dermal Study of Methyl trans-Styryl Ketonea
  Vehicle Control 87.5 mg/kg 175 mg/kg 350 mg/kg
Number weighed at necropsy 10 10 10 10
Necropsy body wt (g) 180 ± 4 174 ± 2 175 ± 3 176 ± 2
Proportion of regular cycling femalesb 10/10 9/10 10/10 10/10
Estrous cycle length (days) 5.0 ± 0.05 4.9±0.08 5.0 ± 0.00 4.9 ± 0.07
Estrous stages (% of cycle)
Diestrus 59.2 57.5 60.0 62.5
Proestrus 17.5 15.0 14.2 15.8
Estrus 20.8 25.8 25.8 20.0
Metestrus 2.5 1.7 0.0 1.7
a  Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the vehicle control group are not significant by Dunnett's test (body weight) or Dunn's test (estrous cycle length). By multivariate analysis of variance, dosed females do not differ significantly from the vehicle control females in the relative length of time spent in the estrous stages. The tests for equality of transition probability matrices among all groups and between the vehicle control group and each dosed group indicated there were no significant differences.
b  Number of females with a regular cycle/number of females cycling
Conclusions:
The study was performed with the related 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. An NOEAL was derived (87.5 mg/kg bw).
Executive summary:

The related 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:
No repeated dose toxicity studies are available for benzalacetone. Therefore, a subchronic study in rats conducted with the related substance methyl-trans-styryl ketone, the nearest analogue, is used for the DNEL derivation and further risk assessment (well-documented NTP report)

Additional information

Repeated dose oral:

The related substance methyl trans-styryl ketone was investigated for its repeated dose toxicity 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).

The related substance methyl trans-styryl ketone 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:

The related substance methyl trans-styryl ketone 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.

The related substance methyl trans-styryl ketone 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 selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study available with the common species(rats) for the related substance methyl-trans-styryl ketone has been used for the DNEL derivation for the oral route of exposure (DNEL oral consumers).

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The study available for rats for the related substance methyl-trans-styryl ketone has been used for the derivation of the dermal and after route to route extrapolation for inhalatory DNEL for workers/consumers.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The study available for rats for the related substance methyl-trans-styryl ketone has been used for the derivation of the dermal and after route to route extrapolation for inhalatory DNEL for workers/consumers.

Justification for classification or non-classification

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