4-hydroxy-2,5-dimethylfuran-2(3H)-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (gavage) male rat fertility study (ICH Guideline 4.1.1, GLP, Key, Rel.1): NOAEL fertility in male rats = 1000 mg/kg bw/day

Effects on fertility for females: no data available

Link to relevant study records

Reference

Endpoint:

fertility, other

Remarks:

Oral male fertility sudy

Type of information:

experimental study

Adequacy of study:

other information

Study period:

16 October 2007 to 6 February 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: ICH Guideline 4.1.1

Deviations:

no

Principles of method if other than guideline:

This oral male fertility study was conducted to determine the potential effects of test item on mating, fertility and gonadal function in male rats. This study included 2 separate mating trials following 2 and 9 weeks of male dose administration (Phases I and II, respectively). The first mating phase, using untreated females, was conducted to detect potential early genotoxic effects on the embryo with reduced risk of test article-related deficiencies in mating or fertility. The second mating phase was conducted following male exposure throughout a complete spermatogenic cycle using a second set of untreated females.

GLP compliance:

yes

Limit test:

no

Justification for study design:

- Basis for dose level selection: Dosage levels were selected based on the results of the 5-day tolerability phase of this study. In this phase, there were no test article-related effects on clinical signs, body weights, body weight changes or food consumption at 1000 mg/kg bw/day (the highest dosage level). Based on these results, the dosage levels selected for administration to male rats for the fertility phase of the study were 0, 100, 500 and 1000 mg/kg bw/day.
- Route of administration: The selected route of administration for this study was oral (gavage) because this is the main intended route of exposure for humans.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproduction studies per ICH Guideline 4.1.1. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age and weight at study initiation: At the initiation of dose administration (study day 0), the males were approximately 9 weeks old; body weights ranged from 256 to 333 g. Males were approximately 11 and 18 weeks old at the initiation of mating with Phase I and II females, respectively. The Phase I females were approximately 12 weeks old when paired with the males on study day 13; body weights ranged from 217 to 288 g on gestation day 0. The Phase II females were approximately 12 weeks old when paired with the males on study day 62; body weights ranged from 226 to 283 g on gestation day 0.
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, stainless steel, wire-mesh cages suspended above cage board. The cage-board was changed at least 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the animals were returned to individual cages. Nesting material was not required, as euthanasia was scheduled prior to the date of expected parturition.
- Diet: Basal diet (PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002), ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: The males were housed for an acclimation period of 12 days prior to the first day of treatment. The phase I and phase II females were housed for an acclimation period of 12 days through the first day of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 70.8-73.9 °F (21.5-23.3 °C)
- Humidity: 38.1-50.2%
- Air changes: Air handling units were set to provide a minimum of 10 fresh air changes per hour.
- Photoperiod: 12 hour light / 12 hour dark photoperiod

IN-LIFE DATES: 16 October 2007 to 6 February 2008

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle, propylene glycol, was transferred to an appropriate sized glass container approximately weekly for administration to the control group males (Group 1) and for preparation of the test article formulations; aliquots were prepared for daily dispensation to the control group. The vehicle was mixed throughout preparation, sampling and dose administration procedures. Dosing formulations for the fertility phase were prepared at the test article concentrations of 10, 50 and 100 mg/mL.
The appropriate amount of the test article for each formulation was weighed into a calibrated glass container. Approximately 70% of the vehicle was added to each container, and the formulations were mixed until uniform using a magnetic stirrer. Vehicle was added to each container to bring the formulations to the calibration mark. The test article formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored refrigerated, protected from light. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures. The test article formulations were visually inspected by the Study Director on 28 October 2007, and were found to be visibly homogeneous and acceptable for dose administration.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not reported
- Concentration in vehicle: 10, 50 and 100 mg/mL
- Source: Spectrum Quality Products, Inc., New Brunswick, New Jersey.
- Lot/batch no. : SQ0397, US0803, UQ1105, WS0076 and WT0050, exp. dates: 26 October 2010, 8 November 2010, 10 November 2010, 27 April 2009 and 4 June 2009, respectively
- Purity: not reported
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: Animals were paired on a 1:1 basis within each treatment group.
- Length of cohabitation: Two mating trials were conducted in this study. For the first mating trial, the treated males were paired with untreated Phase I females following 14 days of treatment. In the second mating trial, the same treated males were paired with a second set of untreated Phase II females following 63 days of treatment. Each female was housed in the home cage of the male.
- For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day. Male pre-coital intervals were based on the total number of days a male was paired with a female.
- Proof of pregnancy: Presence of a copulatory plug or the presence of sperm in a vaginal lavage referred to as gestation day 0.
- If no evidence of copulation was observed after 7 days, a new female (not previously assigned to the study) was arbitrarily assigned and placed with the male for an additional 7 days. If evidence of copulation was not detected after 7 days of pairing, any females that had not shown evidence of mating were placed in suspended wire-mesh cages, and remained there until euthanasia.
- After successful mating each pregnant female was caged (how): Following positive evidence of mating, the animals were returned to individual cages.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the initiation of dose administration, duplicate samples (1 mL each) for homogeneity determination were collected on 15 October 2007 from the top, middle and bottom strata of the 10 and 100 mg/mL non-dosing formulations. In addition, duplicate samples (1 mL each) for stability and resuspension homogeneity determinations were collected on 25 October 2007 from the top and bottom strata of aliquots from these same dosing suspensions following refrigerated storage, protected from light for 10 days. Samples (1 mL each) for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for the first and twelfth weeks of dose administration for the fertility phase. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC using a validated gas chromatography method with flame ionization detection.

Duration of treatment / exposure:

Males received 14 daily doses prior to mating with untreated Phase I females and 63 daily doses prior to mating with untreated Phase II females. Males were dosed throughout both mating periods through 1 day prior to euthanasia for a total of 91 93 doses. The females were not dosed.

Frequency of treatment:

Once daily

Details on study schedule:

Not applicable

Dose / conc.:

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

Remarks:

Group 1 (control)

Dose / conc.:

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

Remarks:

Group 2

Dose / conc.:

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

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4

No. of animals per sex per dose:

Males: 25 males/dose
Females (not dosed on study but mated with treated males)
Phase I females: 25, 27*, 25 and 27* females mated with males dosed at 0, 100, 500, 1000 mg/kg bw/day, respectively
Phase II females: 30*, 26*, 27* and 25 females mated with males dosed at 0, 100, 500, 1000 mg/kg bw/day, respectively
* Following no evidence of copulation during the first 7 days of mating, new females (not previously assigned to the study) were placed with the males for an additional 7 days.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of the 5-day tolerability phase of this study. Animals were administered the test item, in the vehicle propylene glycol, orally by gavage once daily during study days 0-4 at dosage levels of 0, 500 and 1000 mg/kg bw/day. In this phase, there were no test article-related effects on clinical signs, body weights, body weight changes or food consumption at 1000 mg/kg bw/day (the highest dosage level). Based on these results, the dosage levels selected for administration to male rats for the fertility phase of the study were 0, 100, 500 and 1000 mg/kg bw/day.
- Rationale for animal assignment: At the conclusion of the respective acclimation periods, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements (250-450 g for males, 170-287 g for Phase I females and 170-278 g for Phase II females) was selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into the WIL Toxicology Data Management System (WTDMS™). A printout containing the animal numbers, corresponding body weights and individual group assignments was generated based on body weight stratification randomized in a block design. The animals then were arranged into groups according to the printout.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded daily (prior to test article administration during the treatment period) for males. Each male was also observed for signs of toxicity at the time of (within 15 minutes) and approximately 1 hour following dose administration. Clinical observations regarding general appearance and behavior were recorded weekly for untreated females.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded daily from the first day of treatment, throughout the study and prior to the scheduled euthanasia. Mean daily body weights and body weight changes were presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating treatment periods (study days 0-13 and 0-62) and for the entire treatment period (study days 0-92). When body weights could not be determined for an animal during a given interval (due to an unscheduled death), group mean values were calculated for that interval using the available data. Individual female body weights were recorded once prior to mating and on gestation days 0 and 15. Mean gestation body weights and body weight gains are presented for these intervals.

FOOD CONSUMPTION: Yes
- Individual male food consumption was recorded weekly from the first day of treatment through euthanasia. Food consumption was reported as g/animal/day and g/kg/day for each week. When food consumption could not be determined for an animal during a given interval (due to an unscheduled death, weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data. Food consumption was not recorded during the breeding periods as the animals were cohabited during this time. Food consumption was not recorded for the females.

Oestrous cyclicity (parental animals):

Non applicable

Sperm parameters (parental animals):

Spermatogenic Endpoint Evaluations:
Immediately upon euthanasia, the reproductive tract of each male was exposed via a ventral mid line incision. The right epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate buffered saline (maintained at approximately 37 ºC) with 10 mg/mL bovine serum albumin (BSA). After a 10 minute incubation period, a sample of sperm was loaded into a 100 µm cannula for determination of sperm motility. Because sperm motility can be affected by temperature shock, all cannulas and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37 ºC) using the Hamilton Thorne HTM IVOS computer-assisted sperm analysis (CASA) System Version 12.3.
Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) sperm was reported:
Percent Motile Sperm = (Number of Motile Sperm / Total Number of Sperm Counted) x 100
The right epididymis was then placed in Bouin’s solution for possible microscopic examination. Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique (Linder et al., 1992). Abnormal forms of sperm (double heads, double tails, microcephalic or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
The left testis and epididymis from each male were weighed, stored frozen, homogenized and analyzed for determination of homogenization-resistant spermatid count and calculation of sperm production rate (Blazak et al., 1985) using the Hamilton Thorne CASA system. An aliquot of each sample was added to a solution containing a DNA specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20 µm chamber depth. Illumination from a xenon lamp within the HTM IVOS analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample. The sperm production rate was calculated as follows:
Sperm Production Rate = No. of sperm per gram of testis / 6.1 Days
* 6.1 Days = The rate of turnover of the germinal epithelium

Litter observations:

Non applicable

Postmortem examinations (parental animals):

SACRIFICE
- All animals were euthanized by carbon dioxide inhalation. The females with evidence of mating were euthanized on gestation day 15. The females without evidence of mating were euthanized 8 days following completion of the cohabitation period, and the males were euthanized following completion of post mortem examination of the Phase II females with evidence of mating (day 15 following completion of the second mating trial).

GROSS NECROPSY
- A gross necropsy was performed for the males that died or were euthanized in extremis, the males that survived to the scheduled necropsy and females with no evidence of mating. The necropsy included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord and the thoracic, abdominal and pelvic cavities including viscera.
- All females with evidence of mating were euthanized on gestation day 15. The thoracic, abdominal and pelvic cavities were opened by a ventral mid line incision and the contents were examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The uterus was opened, and the number of corpora lutea on each ovary was recorded. The number and location of all embryos, early resorptions and the total number of implantation sites were recorded. Viability of the embryos was determined with the aid of a dissecting stereomicroscope, if necessary. The individual uterine distribution was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to right distal uterine horn. The carcasses of the dams and all products of conception were then discarded.

ORGAN WEIGHTS
- The following organs were weighed from all males at the scheduled necropsy:
Brain, Epididymides# (total and cauda), Pituitary gland and Testes#
# These paired organs were weighed separately.
Organ to final body weight and organ to brain weight ratios were calculated.

HISTOPATHOLOGY
- At the time of necropsy, the following tissues and organs were collected from the males and placed in 10% neutral buffered formalin except as noted below:
Coagulating glands (2), Pituitary gland, Prostate gland, Seminal vesicles (2), Testis with epididymis and vas deferens (1)*, All gross lesions**
* = Testis and epididymis (right only) were fixed in Bouin’s solution. For males that died or were euthanized in extremis, both testes and epididymides were fixed in Bouin’s solution.
** = Representative sections of corresponding organs from a sufficient number of controls were retained for comparison.
The uteri of females without macroscopic evidence of implantation were opened and placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

UNSCHEDULED DEATHS
A detailed gross necropsy was performed on each male that died or was euthanized in extremis during the course of the study and the cause of death was recorded, if possible. Gross lesions were saved and representative sections of corresponding organs from a sufficient number of controls were retained for comparison. Sperm analysis was not performed and organ weights were not collected. All carcasses were discarded.

Postmortem examinations (offspring):

Non applicable

Statistics:

See "Any other information on materials and methods incl. tables".

Reproductive indices:

Mating, fertility and copulation indices were calculated as follows:
Male Mating Index (%) = (No. of Males with Evidence of Mating (or Females Confirmed Pregnant) / Total No. of Males Used for Mating) x 100
Male Fertility Index (%) = (No. of Males Siring a Litter / Total No. of Males Used for Mating) x 100
Male Copulation Index (%) = ((No. of Males Siring a Litter) / No. of Males with Evidence of Mating (or Females Confirmed Pregnant)) x 100

Group Mean Litter Basis:
Postimplantation Loss/Litter = (No. Dead Embryos, Resorptions (Early)/Group) / (No. Gravid Females/Group)

Proportional Litter Basis:
Summation Per Group (%) = (Sum of Postimplantation Loss/Litter (%)) / (No. Litters/Group)
Where:
Postimplantation Loss/Litter (%) = [(No. Dead Embryos, Resorptions (Early)/Litter) / (No. Implantation Sites/Litter)] x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treated males:
Test article-related clinical observations were noted with increased incidence and/or severity in the 100, 500 and 1000 mg/kg bw/day group males compared to the control group at the time of or approximately 1 hour following dose administration. Salivation-related findings (including salivation prior to dosing and clear material around the mouth, nose and/or forelimbs) occurred at all dosage levels beginning as early as study day 5 and generally persisted throughout the entire treatment period. As a result, an increased incidence of hair loss around the mouth was noted in the 1000 mg/kg bw/day group males during the daily examinations. Evidence of salivation prior to dose administration was attributed to the taste of the test article and was not considered evidence of systemic toxicity.

Untreated females:
Clinical findings were noted similarly in all groups of untreated females and were findings commonly
observed in laboratory rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treated males:
There was no test article-related mortality/moribundity noted in this study. One male (no. 85441) in the 1000 mg/kg bw/day group was euthanized in extremis on study day 61. Clinical observations noted for this male on the day prior to and/or day of euthanasia included moderate to severe yellow material around the urogenital and ventral abdominal areas. Subsequently, the attending veterinarian diagnosed this male with urolithiasis, as corroborated by necropsy findings of depressed areas on the kidneys, cystic kidney, dilated pelvis, distended ureters and urinary bladder and urinary bladder calculi. Therefore, the moribund condition of this male was not attributed to test article administration. In addition, male no. 85420 in the control group was found dead on study day 70; there were no remarkable clinical observations noted prior to death. Macroscopic findings noted at necropsy included dark red discoloration of the lungs, lungs not fully collapsed and red matting around the nasal and buccal areas. All other males survived to the scheduled necropsy.

Untreated females:
All females in Phase I and Phase II survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treated males:
- Mean male body weight gain in the 1000 mg/kg bw/day group was similar to the control group prior to the first mating phase (study days 0-13); however, mean body weight gains tended to be slightly lower than the control group throughout the remainder of the treatment period. The differences were of sufficient magnitude to result in an overall lower (statistically significant, 224g and 255g in male rats dosed at 1000 mg/kg bw/day and control rats, respectively) mean body weight gain when the entire treatment period (study days 0-92) was evaluated, and mean body weight was up to 5.7% lower (not statistically significant) than the control group by the end of the treatment period. This slight decrease in mean body weight gain, in the absence of any corresponding decrements in food consumption, was considered test article-related but not an adverse sign of systemic toxicity.
- Mean body weights and body weight gains were unaffected by test article administration in the 100 and 500 mg/kg bw/day groups. Differences from the control group occasionally achieved significance (p<0.05 or p<0.01) but generally did not occur in a dose-related manner and no trends were apparent.

Untreated females:
Mean maternal body weights and body weight gains were similar among all groups of untreated
females throughout gestation.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 100, 500 and 1000 mg/kg bw/day groups was unaffected by the test article during the treatment period. The values in the test article treated groups were generally similar to the control group values. Differences from the control group were slight, did not occur in a dose related manner and/or were not statistically significant.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Test article-related behavioral findings of subdued appearance upon cage-side observation only (when the animals were relaxed, their heads were resting on the bottom of the cage or food jar and the animals appeared hypoactive, but exhibited normal behavior when stimulated) were noted in the 500 and 1000 mg/kg bw/day groups approximately 1 hour following dose administration between study days 5 and 82, but did not persist to the next daily observation. In addition, rocking, lurching, or swaying while ambulating was noted for 7 males in the 1000 mg/kg bw/day group primarily during study days 4-12; however, this higher incidence was attributed to a single male, with the other 6 males in this group exhibiting only a single occurrence. Moreover, this finding was also noted in the control group and did not persist to the next daily examination. Therefore, these behavioral findings were not considered adverse.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

- No test article-related effects were observed in spermatogenesis endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility and morphology) at 100, 500 and 1000 mg/kg bw/day.
- Mean epididymal sperm concentration in the 1000 mg/kg bw/day group (330.3 million/gram) was slightly lower than the control group value (394.2 million/gram); the difference was not significant, but was below the minimum mean value in the WIL reproductive historical control data (339.1 million/gram). This finding was primarily attributed to 1 male (no. 85394) with atypically small or underdeveloped reproductive organs and with an epididymal sperm concentration of 8.9 million/gram.
- The percent of morphologically normal sperm in the 500 and 1000 mg/kg bw/day groups (96.2% and 94.6%, respectively) was slightly lower than the control group (98.4%), primarily due to the percentages of sperm with normal shaped heads without flagella (1.7% and 3.0%, respectively) or normal flagella without heads (2.0% and 2.4%, respectively) compared to the control group (0.9% and 0.7%, respectively). Differences from the control group were not significant at either dose level, but the values at 1000 mg/kg bw/day were outside the WIL reproductive historical control data ranges (96.1%-99.1%, 0.1%-1.8% and 0.0% 2.3%, respectively). The slightly lower incidences of morphologically normal sperm at 500 and 1000 mg/kg bw/day were attributed to single males in each of these groups with atypically high numbers of morphologically abnormal sperm (separated head and flagellum; 54.5% and 56.5%, respectively). When the mean percentage of morphologically normal sperm was recalculated at 1000 mg/kg bw/day, without considering this 1 male, the mean percentage of normal sperm increases to 96.9%, which is within the WIL historical control data range. Moreover, there were no corresponding effects on other spermatogenic parameters or male reproductive performance in the 1000 mg/kg bw/day group; therefore, the slightly lower mean percentage of morphologically normal sperm relative to the control group was not attributed to the test article.

Reproductive performance:

no effects observed

Description (incidence and severity):

Treated males:
- No test article-related effects on male reproductive performance (male mating, fertility and copulation) or pre-coital intervals were observed at 100, 500 and 1000 mg/kg bw/day when males were mated with Phase I or Phase II females.
- The male fertility and copulation indices in the 1000 mg/kg bw/day group were slightly lower than the concurrent control group values for mating Phase I. However, the differences from the concurrent control group were not statistically significant, were within the WIL reproductive historical control data ranges and were not reproduced in the second mating phase (following a longer treatment period). During mating Phase I, 1, 1, 0 and 4 males in the control, 100, 500 and 1000 mg/kg bw/day groups, respectively, did not sire a litter. With the exception of 1 male (no. 85394) in the 1000 mg/kg bw/day group (with atypically small or underdeveloped reproductive organs), all of these males sired a litter in the Phase II breeding trial. During mating Phase II, 1 male each in the control, 100, 500 and 1000 mg/kg bw/day groups did not sire a litter.

Untreated females:
Intrauterine survival of the embryos was unaffected by administration of the test article to the males at dosage levels of 100, 500 and 1000 mg/kg bw/day when paired following 14 daily doses (Phase I) or following 63 daily doses (Phase II). No statistically significant differences from the control group were noted. Intrauterine parameters evaluated included mean litter proportions of pre- and postimplantation losses and mean numbers of corpora lutea, implantation sites and viable embryos.

Key result

Dose descriptor:

NOAEL

Remarks:

male reproductive / systemic toxicity

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

reproductive function (sperm measures)

reproductive performance

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 7.8.1/1: Results of Homogeneity, Stability and Resuspension Homogeneity Analyses

Analyses	Group 2 (10 mg/mL)	Group 4 (100 mg/mL)
Homogeneity Assessment of the 15 October 2007 Formulations
Mean Concentration (mg/mL)	9.94	96.1
RSD (%)	2.2	1.5
Mean % of Target	99.4	96.1
10-Day Refrigerated Resuspension Homogeneity and Stability Assessment of the 15 October 2007 Formulations
Mean Concentration (mg/mL)	9.77	99.1
RSD (%)	0.67	0.24
Mean % of Target	97.7	99.1
Mean % of Time Zero	98.3	103

 

Table 7.8.1/2: Results of Concentration Analyses

Date of Preparation	Mean Concentration, mg/mL (% of Target)
	Group 2 (10 mg/mL)	Group 3 (50 mg/mL)	Group 4 (100 mg/mL)
26 October 2007	11.0 (110)	54.3 (109)	109 (109)
10 January 2008	10.1 (101)	50.3 (101)	98.8 (98.8)

 

Based on these results, the analyzed dosing formulations were within the WIL Research Laboratories, LLC’s standard operating procedure’s range for suspensions (85% to 115% of target concentration), were homogeneous and were stable following 10 days of refrigerated storage. Therefore, the protocol-specified dosages of test article were administered to the animals. Test article was not detected in the vehicle formulations used for the control group (Group 1).

Conclusions:

Under the test conditions, the reproductive NOAEL in male rats was considered to be 1000 mg/kg bw/day based on the absence of effects on spermatogenic parameters, organ weights, reproductive performance and embryonic survival. Slight decreases in body weight gain at 1000 mg/kg bw/day in the absence of an effect on food consumption, was not considered adverse; therefore, the NOAEL for male systemic toxicity was considered to be 1000 mg/kg bw/day in rats.

Executive summary:

A male fertility study was conducted to determine the potential effects of test item on mating, fertility and gonadal function in male rats. Test item was administered orally by gavage once daily to 3 groups of 25 male Crl:CD(SD) rats. Dosage levels were 100, 500 and 1000 mg/kg bw/day administered at a dosage volume of 10 mL/kg bw/day. A concurrent control group of 25 males received the vehicle (propylene glycol) on a comparable regimen. The choice of doses was based on a preliminary 5-day tolerability phase at 0, 500 and 1000 mg/kg bw/day. In the main study, males were approximately 9 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating with untreated Phase I females and 63 daily doses prior to mating with untreated Phase II females. Males were dosed throughout both mating periods through 1 day prior to euthanasia for a total of 91‑93 doses. The females were not dosed. All animals were observed twice daily for mortality and moribundity. Clinical observations and body weights were recorded at appropriate intervals for all animals; food consumption was recorded only for the treated males. A laparohysterectomy was performed on gestation day 15 for each female with evidence of mating. All males were euthanized following post mortem examination of the Phase II females. Spermatogenic evaluations were conducted on all males at the time of necropsy. Selected organs from all surviving males were weighed and/or retained.

There was no test item-related mortality/moribundity noted in this study. Salivation‑related findings (clear material around the mouth, nose and/or forelimbs and salivation prior to dosing) occurred in a dose‑related manner within 15 minutes of and 1 hour following dose administration; these findings were likely due to the taste of the test item and not considered adverse. Additionally, test item-related behavioral findings consisting of rocking, lurching or swaying while ambulating at 1000 mg/kg bw/day and subdued appearance upon cage side observation at 500 and 1000 mg/kg bw/day were noted approximately 1 hour following dose administration. These behavioral findings did not persist to the next daily examination, occurred at very low incidence (primarily 1 occurrence in individual animals) and was also noted in the control group (rocking, lurching, or swaying); therefore, these findings were not considered adverse.

A slightly lower mean body weight gain was noted in the 1000 mg/kg bw/day group when evaluated for the overall treatment period. As a result, mean body weight was slightly lower on study day 92. The effects on body weight noted at 1000 mg/kg bw/day were minimal and occurred in the absence of an effect on food consumption; therefore, this finding was not considered adverse. Mean body weights, body weight gains and food consumption in the 100 and 500 mg/kg bw/day groups were similar to the control group throughout the treatment period. 

No test item-related effects on male reproductive performance (Mating, fertility and copulation indices) were observed at 100, 500 and 1000 mg/kg bw/day when males were mated with Phase I (following 14 days of treatment) or Phase II (following 63 days of treatment) females. Furthermore, there were no test item-related effects on spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility and morphology, reproductive organs or macroscopic findings) at 100, 500 and 1000 mg/kg bw/day. The mean percentage of sperm with abnormal morphology (separated head and flagellum) was slightly higher in the 500 and 1000 mg/kg bw/day groups; however, this was primarily attributed to a single male in each of these groups with atypically high percentages of morphologically abnormal sperm. In the absence of any other effects on reproductive performance or corresponding effects on other spermatogenic parameters, this finding was not considered test item‑related.

 

Under the test conditions, the reproductive NOAEL in male rats was considered to be 1000 mg/kg bw/day based on the absence of effects on spermatogenic parameters, organ weights, reproductive performance and embryonic survival. Slight decreases in body weight gain at 1000 mg/kg bw/day in the absence of an effect on food consumption, was not considered adverse; therefore, the NOAEL for male systemic toxicity was considered to be 1000 mg/kg bw/day in rats.

Effect on fertility: via oral route

Quality of whole database:

A study, GLP-compliant and of high quality (Klimisch score = 1), was identified. However, this study only assess the effects of the substance on male rats fertility. No data was available on the effects on the fertility of females. Therefore this study is not sufficient to cover the endpoint. However no further information is necessary at annex VII level.

Additional information

A study was identified (WIL, 2008, Rel.1). This male fertility study was conducted to determine the potential effects of test item on mating, fertility and gonadal function in male rats. Test item was administered orally by gavage once daily to 3 groups of 25 male Crl:CD(SD) rats. A concurrent control group of 25 males received the vehicle (propylene glycol) on a comparable regimen.Male rats were approximately 9 weeks of age at the beginning of test item administration. They received 14 daily doses prior to mating with untreated Phase I females and 63 daily doses prior to mating with untreated Phase II females. The male rats were dosed throughout both mating periods through 1 day prior to euthanasia for a total of 91‑93 doses. The females were not dosed. All animals were observed twice daily for mortality and moribundity. Clinical observations and body weights were recorded at appropriate intervals for all animals; food consumption was recorded only for the treated males. A laparohysterectomy was performed on gestation day 15 for each female with evidence of mating. All males were euthanized following post mortem examination of the Phase II females. Spermatogenic evaluations were conducted on all males at the time of necropsy. Selected organs from all surviving males were weighed and/or retained.

There was no test item-related mortality/moribundity noted in this study. Salivation‑related findings (clear material around the mouth, nose and/or forelimbs and salivation prior to dosing) occurred in a dose‑related manner within 15 minutes of and 1 hour following dose administration; these findings were likely due to the taste of the test item and not considered adverse. Additionally, test item-related behavioral findings consisting of rocking, lurching or swaying while ambulating at 1000 mg/kg bw/day and subdued appearance upon cage side observation at 500 and 1000 mg/kg bw/day were noted approximately 1 hour following dose administration. These behavioral findings did not persist to the next daily examination, occurred at very low incidence (primarily 1 occurrence in individual animals) and was also noted in the control group (rocking, lurching, or swaying); therefore, these findings were not considered adverse.

A slightly lower mean body weight gain was noted in the 1000 mg/kg bw/day group when evaluated for the overall treatment period. As a result, mean body weight was slightly lower on study day 92. The effects on body weight noted at 1000 mg/kg bw/day were minimal and occurred in the absence of an effect on food consumption; therefore, this finding was not considered adverse. Mean body weights, body weight gains and food consumption in the 100 and 500 mg/kg bw/day groups were similar to the control group throughout the treatment period. 

No test item-related effects on male reproductive performance (Mating, fertility and copulation indices) were observed at 100, 500 and 1000 mg/kg bw/day when males were mated with Phase I (following 14 days of treatment) or Phase II (following 63 days of treatment) females. Furthermore, there were no test item-related effects on spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility and morphology, reproductive organs or macroscopic findings) at 100, 500 and 1000 mg/kg bw/day. The mean percentage of sperm with abnormal morphology (separated head and flagellum) was slightly higher in the 500 and 1000 mg/kg bw/day groups; however, this was primarily attributed to a single male in each of these groups with atypically high percentages of morphologically abnormal sperm. In the absence of any other effects on reproductive performance or corresponding effects on other spermatogenic parameters, this finding was not considered test item‑related.

 

Under the test conditions, the reproductive NOAEL in male rats was considered to be 1000 mg/kg bw/day based on the absence of effects on spermatogenic parameters, organ weights, reproductive performance and embryonic survival.Slight decreases in body weight gain at 1000 mg/kg bw/day in the absence of an effect on food consumption, was not considered adverse; therefore, the NOAEL for male systemic toxicity was considered to be 1000 mg/kg bw/day in rats.

This study only assess the effects of the substance on male rats fertility. No data was available on the effects on the fertility of females. Therefore this study is not sufficient to cover the endpoint. However no further information is necessary at annex VII level.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed for the substance according to the Regulation (EC) No 1272/2008 and to the GHS.

However, the available study only assess the effects of the substance on male rats fertility. No data was available on the effects on the fertility of females. Therefore this study is not sufficient to cover the endpoint. However no further information is necessary at annex VII level.