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Endpoint:
toxicity to reproduction
Remarks:
other: 3-month feeding study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This information comes from an NTP-report (TR 572), 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:
other: OECD TG 408
Deviations:
not applicable
Principles of method if other than guideline:
Groups of 10 male and 10 female F344/N rats were fed diets containing 0%, 0.025%, 0.05%, 0.1%, 0.2%, or 0.4% methyl trans-styryl ketone for 14 weeks based on results of a dosed-feed palatability study. Additional clinical pathology groups of 10 male and 10 female rats received the same dosed diets for 24 days. Body weights and clinical findings for rats and mice were recorded initially, weekly, and at the end of the studies. Feed consumption was recorded weekly by cage. Haematology, clinical chemistry, gross and histopathological observations have been performed. At the end of the 3-month feed studies (12 days prior to scheduled terminal sacrifice), samples were collected for sperm motility and vaginal cytology evaluations on rats and mice exposed to 0%, 0.1%, 0.2%, or 0.4%.
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 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: five per cage
- 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: rats: 11 / 12 days of quaratine
Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease. At the end of the studies, sero-logic analyses were performed on five male and five female control rats using the protocols of the NTP Sentinel Animal Program

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 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 formulations were also analysed. Of the dose formulations analysed, 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.
Details on mating procedure:
Not applicable (3 months feeding study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
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 analysed 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:
core animals: 14 weeks;
Additional clinical pathology groups of 10 male and 10 female rats received the same dosed diets for 24 days.
Frequency of treatment:
ad libitum, available in diet for 14 weeks
Details on study schedule:
Not applicable ((3 months feeding study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Remarks:
Doses / Concentrations:
0.025%, 0.05%, 0.1%, 0.2% and 0.4% (corresponding to 18, 36, 72, 145, and 290 mg/kg bw for males and 19, 38, 77, 150, and 300 mg/kg bw for females)
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
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.
Parental animals: Observations and examinations:
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 consumption was recorded weekly by cage.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: rats: on days 4 and 24 and from clinical pathology study rats and from surviving core study animals at study termination
- Anaesthetic used for blood collection: Yes (70% CO2/30% O2 mixture)
- Animals fasted: No data
- How many animals: all surviving core animals (rats) and clinical pathology rats
- 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: rats: on days 4 and 24 and from clinical pathology study rats and from surviving core study rats at study termination.
- Anaestethitc used for blood collection: Yes (70% CO2/30% O2 mixture)
- Animals fasted: No data
- How many animals: all surviving core animals (rats) and clinical pathology rats
- Parameters examined: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts.
Oestrous cyclicity (parental animals):
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%. 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).
Sperm parameters (parental animals):
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 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 hemacytometer.
Litter observations:
Not applicable (3 months feeding study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Postmortem examinations (parental animals):
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.

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.

Postmortem examinations (offspring):
Not applicable (3 months feeding study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose related trends. All reported P values for the survival analyses are two sided. The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Furthermore, an analysis of continuous variables was performed (for references, please refer to NTP report).
Reproductive indices:
Not applicable (3 months feeding study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Offspring viability indices:
Not applicable (3 months feeding study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
diarrhea and hyperactivity in both sexes
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. Feed consumption by exposed and control groups was generally similar.
Food consumption and compound intake (if feeding study):
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. Feed consumption by exposed and control groups was generally similar.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
slight treatment related increased incidences of nephropathy in all exposed groups with an increased severity in the group exposed to 0.4%.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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%.
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
females exposed to 0.4% had a significantly higher probability of extended diestrus than the controls.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All core study rats survived to the end of the study. Treatment-related clinical findings included diarrhea and hyperactivity in both sexes.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
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. Feed consumption by exposed and control groups was generally similar.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
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 (please refer to the table in "Any other information on results incl. tables").

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no significant differences in any of the reproductive organ weights or sperm parameters of male rats at any exposure concentration (please refer to the table in "Any other information on results incl. tables").

ORGAN WEIGHTS (PARENTAL ANIMALS)
No biologically significant organ weight changes were observed in exposed groups of males or females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
In the kidney of male rats, there were slight treatmentrelated 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.

HISTOPATHOLOGY (PARENTAL ANIMALS)
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.

OTHER FINDINGS (PARENTAL ANIMALS)
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. 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, cited in NTP report). No other changes in the hematology or serum chemistry variables were considered attributable to methyl trans-styryl ketone exposure.
Dose descriptor:
NOAEL
Effect level:
290 mg/kg bw/day
Based on:
test mat.
Sex:
male
Basis for effect level:
other: There were no significant differences in any of the reproductive organ weights or sperm parameters of male rats at any exposure concentration.
Remarks on result:
other: Generation: all examined animals (migrated information)
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: all examined animals (migrated information)
Reproductive effects observed:
not specified

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:
Based on the results of sperm motility and vaginal cytology evaluations, the reproductive organ weights, and the histopathology of the reproductive organs, exposure to methyl trans-styryl ketone in feed did not indicate potential for reproductive toxicity in male rats. However, females had significantly higher probabilities of extended diestrus, suggesting that exposure to methyl trans-styryl ketone in feed might produce reproductive toxicity in females.
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%. Goblet cell hyperplasia of the respiratory epithelium of the nose is likely due to inhalation of methyl trans-styryl ketone that volatilized from feed.

Based on the results of sperm motility and vaginal cytology evaluations, the reproductive organ weights, and the histopathology of the reproductive organs, NOEALs for reproductive toxicity is considered to be 290 mg/kg bw, the highest dose level tested, for males and 150 mg/kg bw for females.

Endpoint:
toxicity to reproduction
Remarks:
other: subchronic study
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This information comes from an NTP-report (TR 572), which deals with the related substance: methyl trans-styryl ketone. The quality of this report is considered to be high.
Qualifier:
according to
Guideline:
other: OECD TG 411
Deviations:
not applicable
Principles of method if other than guideline:
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 per day in 95% ethanol, 5 days per week for 14 weeks. 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. Additional clinical pathology groups of 10 male and 10 female rats received the same doses for 23 days. Clinical findings were recorded weekly and at study termination for core study animals. The animals were weighed initially, weekly, and at the end of the study. Hematology and clinical chemistry parameters were analysed in clinical pathology rats on days 4 and 24 and in core study rats at the end of the study. At the end of the study, samples were collected for sperm motility and vaginal cytology evaluations on rats administered 0, 87.5, 175, or 350 mg/kg bw. Necropsies were performed on all core animals. Complete histopathologic examinations were performed on vehicle control rats and on core study 350 mg/kg rats.
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 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: 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
Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease. At the end of the studies, sero-logic analyses were performed on five male and five female control rats using the protocols of the NTP Sentinel Animal Program.

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:
dermal
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 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.
- Concentration (if solution): 95%
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.
Details on mating procedure:
Not applicable (3 months dermal study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
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
Details on study schedule:
Not applicable (3 months dermal study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Remarks:
Doses / Concentrations:
22, 44, 87.5, 175, or 350 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
10
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.
Parental animals: Observations and examinations:
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

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 from clinical pathology group and from core study rats at the end of the studies.
- Anaesthetic used for blood collection: Yes (70% CO2/30% O2 mixture)
- Animals fasted: No data
- How many animals: all animals on study
- 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: on days 4 and 24 from clinical pathology group and from core study rats at the end of the studies.
- Animals fasted: No data
- How many animals: all animals on study
- Parameters examined: urea nitrogen, creatinine, total protein, albumin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and bile salts
Oestrous cyclicity (parental animals):
At the end of the 3-month studies, samples were collected for vaginal cytology evaluations on rats administered 0, 87.5, 175, or 350 mg/kg/bw. 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).
Sperm parameters (parental animals):
At the end of the 3-month studies, samples were collected for sperm motility evaluations on rats administered 0, 87.5, 175, or 350 mg/kg/bw. 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 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 hemacytometer.
Litter observations:
Not applicable (3 months dermal study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Postmortem examinations (parental animals):
SACRIFICE
All surviving animals (at the end of the study)

GROSS PATHOLOGY: Yes
Necropsies were performed on all core study rats. The heart, right kidney, liver, lung, right testis, and thymus were weighed.
HISTOPATHOLOGY: Yes
Complete histopathologic examinations were performed on vehicle control rats and on core study 350 mg/kg rats. 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 kidney, nose, skin, stomach, and uterus were examined in all core study rats.
Postmortem examinations (offspring):
Not applicable (3 months dermal study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose related trends. All reported P values for the survival analyses are two sided. The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess neoplasm and nonneoplastic lesion prevalence. Furthermore, an analysis of continuous variables was performed (for references, please refer to NTP report).
Reproductive indices:
Not applicable (3 months dermal study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Offspring viability indices:
Not applicable (3 months dermal study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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):
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.
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment related lesions of skin; significantly increased incidences of goblet cell hyperplasia of the respiratory epithelium in 350 mg/kg males and 22, 175 and 350 mg/kg females.
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no significant differences in the estrous cyclicity of female rats, at any dose when compared to the vehicle controls (please refer to the table in "Any other information on results incl. tables").

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no significant differences in sperm parameters of male rats, at any dose when compared to the vehicle controls (please refer to the table in "Any other information on results incl. tables").

ORGAN WEIGHTS (PARENTAL ANIMALS)
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.
There were no significant differences in any of the reproductive organ weights at any dose when compared to the vehicle controls.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No effects

HISTOPATHOLOGY (PARENTAL ANIMALS)
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 (PARENTAL ANIMALS)
There were no changes in the hematology or serum chemistry variables attributable to methyl trans-styryl ketone administered dermally to rats for 14 weeks.
Dose descriptor:
NOAEL
Effect level:
350 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 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.
Remarks on result:
other: Generation: animals on study (migrated information)
Not applicable (3 months dermal study with a screening of sperm motility, vaginal cytology and gross and histopathological observations of reproductive organs).
Reproductive effects observed:
not specified

Table: 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: 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:
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. Based on the results of this study, NOAEL of 350 mg/kg bw for rats is considered for reproductive toxicity.
Executive summary:

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. There were no significant differences in any of the reproductive organ weights. 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. Based on the results of this study, NOAEL of 350 mg/kg bw for rats is considered for reproductive toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two 3-month studies available (feeding study and dermal study, each in rats and in mice) for the structural analogue methyl trans-styryl ketone. The overall quality of the database is considered to be high.
Effect on fertility: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
350 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two 3-month studies available (feeding study and dermal study, each in rats and in mice) for the structural analogue methyl trans-styryl ketone. The overall quality of the database is considered to be high.
Additional information

Sub-chronic Feed Studies

Rat

The related substance methyl trans-styryl ketone was investigated for its repeated dose toxicity via the oral route (NTP, 2012a). Groups of 10 male and 10 female F344/N 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 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%. Goblet cell hyperplasia of the respiratory epithelium of the nose is likely due to inhalation of methyl trans-styryl ketone that volatilized from feed.

Based on the results of sperm motility and vaginal cytology evaluations, the reproductive organ weights, and the histopathology of the reproductive organs, NOEALs for reproductive toxicity is considered to be 290 mg/kg bw, the highest dose level tested, for males and 150 mg/kg bw for females.

Mouse

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 B6C3F1 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%. Goblet cell hyperplasia of the respiratory epithelium of the nose is likely due to inhalation of methyl trans-styryl ketone that volatilized from feed.

Based on the results of sperm motility and vaginal cytology evaluations, the reproductive organ weights, and the histopathology of the reproductive organs, NOAEL for reproductive toxicity is considered to be 750 mg/kg bw, the highest dose level tested, for males. No NOAEL could be identified for females due to the extended diestrus at the dose levels that were chosen for the evaluation of reproductive toxicity.

Sub-chronic Dermal Studies

Rat

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 (NTP, 2012a). 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. There were no significant differences in any of the reproductive organ weights. 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.

Based on the results of this study, NOAEL of 350 mg/kg bw for rats is considered for reproductive toxicity.

Mouse

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 (NTP, 2012a). 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 no significant differences in any of the reproductive organ weights. There were treatment-related increased incidences of epidermal hyperplasia, hyperkeratosis, 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. Based on the results of this study, NOAEL of 350 mg/kg bw for mice is considered for reproductive toxicity.


Short description of key information:
There was no indication based upon the sperm motility and vaginal cytology evaluation results, reproductive organ weights, and histopathology of the reproductive organs that methyl trans-styryl ketone has any potential to be a reproductive toxicant in male or female rats or mice when applied dermally. Since significant amounts of the test chemical were absorbed dermally in rats and in mice, the dermal route of administration would provide a sufficient amount of chemical systemically available to assess possible toxicity in tissues, including in reproductive organs (Sauer et al., 1977; NTP, 2012a).

Justification for selection of Effect on fertility via oral route:
Only one study in rats available on oral route of exposure.

Justification for selection of Effect on fertility via dermal route:
Only one study in rats available on dermal route of exposure.

Effects on developmental toxicity

Description of key information
Benzalacetone was predicted to be negative for developmental toxicity. Structural analogue chemicals identified with the aid of the OECD QSAR Toolbox produced developmental effects either at the dose levels of maternal toxicity or did not induce signs of developmental toxicity at all.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
78.8 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Read-across from structurally similar substances with the similar mechanistic profiling results.
Additional information

There are no data on developmental toxicity of benzalacetone. Therefore, struturally similar chemicals with data on developmental toxicity have been searched for benzalacetone with the aid of the OECD QSAR Toolbox v3.1. The substance was not categorized into OECD HPV Chemical Categories or US EPA New Chemical Categories. However, the substance is assigned as "Vinyl/Allyl ketone" according to the "Aquatic Toxicity Classification by ECOSAR". Therefore chemicals with the same classification have been retrieved from the Toolbox. Two predictions have been run using units either log (1/mol/kg/day) or mg/kg bw/day.

According to the profiling results, benzalacetone binds to proteins via Michael addition mechanism. According to the profiling method "Protein binding by OASIS v1.1, the identified mechanism of binding was "Michael addition on conjugated systems with electron withdrawing group >> alpha,beta-carbonyl compounds with polarized double bonds". According to the profiling method "Protein binding by OECD" the identified mechanism of binding was "Michael addition for polarised alkene-ketones". Since protein binding can be relevant for developmental toxicity, the chemicals which do not bind to proteins or possess other mechanisms of protein binding, were eliminated. Thereafter, the chemical category was refined eradicating chemicals with halogens in their structure. Subcategorisation according to the "Organic functional groups" destroyed the category: there were no substances with the same organis functional groups.

The first prediction is basedon the data of two structurally similar chemicals (CAS 499 -44 -5 and CAS 32388 -55 -9), which are not developmental toxicants. In the developmental studies, the both chemicals produced developmental effects at the dose levels of maternal toxicity.

The second prediction is based on the data of only one chemical (CAS 79 -77 -6). Benzalacetone and the analogue chemical both possess the same 3-buten-2-one moiety. However, benzalacetone contains a benzene ring while the analogue chemical has a cyclohexene structure. Both chemicals bind to proteins. They are highly reactive to cysteine peptide depletion and are assigned to "alpha, beta-carbonyl compounds with polarized multiple bonds" by the profiling method "DPRA Cysteine Peptide Depletion". The potency of protein binding of both chemicals is moderate. However, their mechanisms of protein binding are different according to the two profiling methods (OASIS and OECD). In the developmental study according to the OECD 414, the analogue chemical induced no adverse signs of developmental toxicity.


Justification for selection of Effect on developmental toxicity: via oral route:
No study is selected since both predictions are of equal value.

Justification for classification or non-classification

Due to the fact that there were no adverse effects on reproductive organs and tissues as well as no impact on sperm parameters or estrous cyclicity in rats and in mice if applied dermally (the relevant route of exposure) during 3 month, the test material does not meet criteria for classification and labelling according to the Eurpean Regulation (EC) 1272/2008.