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Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
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

Test material

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

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals 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

Administration / exposure

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).
Doses / concentrations
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.

Examinations

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).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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

Details on results (P0)

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.

Effect levels (P0)

open allclose all
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)

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.