Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study is conducted according to guidelines and GLP.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): BIOBAN™ CS-1246 Antimicrobial
- Physical state: Pale yellow liquid
- Analytical purity: The purity of the test material was determined to be 98.7% on an anhydrous basis by gas chromatography-flame ionization detection. Karl Fischer titration of the test material determined that it contained 0.30% water. Identification was done by Fourier Transform-infrared spectroscopy and gas chromatography-mass spectrometry.
- Lot/batch No.: TC1031LAH3
- Stability under test conditions: BIOBAN™ CS-1246 was found to be stable in corn oil for at least 25 days at concentrations ranging from 0.025 to 25%. Dose solutions for the current study were prepared and used within the stability limits.
- Storage condition of test material:
- Concentration and homogeneity: Analyses of all dose levels and control to determine concentration of the test material were conducted at least four times (at study start, P1 mating, P1 lactation and P2 pre-mating). The homogeneity of the low- and high-dose levels was determined concurrent with dose confirmation. The analyses were conducted using gas chromatography-mass spectrometry (GCMS) with internal and external standards.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: Approximately six weeks at initiation of treatment.
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Housing: After assignment to study, animals were housed singly in stainless steel cages, except during breeding (one male and one female) and during the littering phases of the study. During littering, dams (and their litters) were housed in plastic cages provided with ground corncob nesting material from approximately GD 19 until completion of lactation. Cages had wire mesh floors and were suspended above catch pans. Nonwoven
gauze was placed in the cages to provide a cushion from the flooring for rodent feet. The gauze also provided environmental enrichment. In order to better visualize plugs, gauze was not placed in cages during the breeding phase. Cages contained a feed crock and a pressure activated lixit valve-type watering system
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (ad libitum): Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form.
- Water (ad libitum): Muncipal water was provided ad libitum.
- Acclimation period: at least one week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod: (12hrs dark /12 hrs light)

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: Not applicable
Vehicle:
corn oil
Details on exposure:
Rats were exposed for 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations.
Dosing solutions were prepared by mixing the test material in corn oil at concentrations of 1.25, 6.25, or 37.5 mg/ml and administered at a dose volume of 4 ml/kg to achieve the targeted dose levels. Dose solutions were not adjusted for purity. Dose volumes were adjusted at least weekly based on the most recent individual body weights. The control rats were dosed with corn oil at 4 ml/kg body weight. F1 offspring assigned to the P2 generation began oral gavage dosing on PND 22. F1 offspring selected for necropsy were not directly dosed via gavage. Dose solutions were prepared periodically throughout the study based upon stability data.
Details on mating procedure:
Breeding of the P1 adults commenced after approximately 10 weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating occurred or two weeks elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were separated from the male and returned to their home cage.

If a breeding male died or was removed from study, a substitute partner (from the same dose group) that already completed mating was provided. If mating did not occur after two weeks, the animals were separated without further opportunity for mating.

Approximately 10 weeks after all F1 litters have been weaned, F1 offspring randomly selected to become P2 adults were bred as described above.

Cohabitation of male and female littermates was avoided. A second breeding of the P1 or P2 adults was not conducted.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of all dose levels and control to determine concentration of the test material were conducted at least four times (at study start, P1 mating, P1 lactation and P2 pre-mating). The homogeneity of the low- and high-dose levels was determined concurrent with dose confirmation. The analyses were conducted using gas chromatography-mass spectrometry (GCMS) with internal and external standards.
BIOBAN™ CS-1246 was found to be stable in corn oil for at least 25 days at concentrations ranging from 0.025 to 25%. Dose solutions for the current study were prepared and used within the stability limits.
Duration of treatment / exposure:
Exposure period: 10 wk pre-breeding, mating, gestation, lactation through 2 generations
Premating exposure period: Male: 10 weeks; Female: 10 weeks
Frequency of treatment:
Frequency of treatment: Once daily
Details on study schedule:
Groups of 27 male and 27 female Crl:CD(SD) rats were administered the test material seven days/week via oral gavage in corn oil at dose levels of 0, 5, 25, and 150 mg BIOBAN™ CS-1246/kg of body weight/day for approximately ten weeks prior to breeding, and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations to evaluate the potential for reproductive toxicity and effects on neonatal growth and survival. A comprehensive evaluation of male and female reproductive systems was conducted and included an evaluation of estrous cyclicity, gonadal function, mating performance, conception, gestation, parturition, lactation as well as survival, growth and development off the offspring. In-life observations, body weights, feed consumption and litter data were evaluated. In addition, a gross necropsy of the P1 and P2 adults were conducted with extensive histopathologic examination of reproductive organs and target tissues. Test material administration began on December 15, 2006 and was continued up until the day prior to necropsy. The F1 weanlings were necropsied from April 09, 2007 to April 19, 2007. The P1 adults were necropsied from April 23, 2007 to April 26, 2007 (males) and April 30, 2007 to May 01, 2007 (females). The F2 weanlings were necropsied from August 14, 2007 to August 22, 2007. The P2 adults were necropsied from August 23, 2007 to August 24, 2007 (females) and August 27,
2007 to August 30, 2007.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
25 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
27
Control animals:
yes, concurrent vehicle
Details on study design:
Groups of 27 male and 27 female Crl:CD(SD) rats were obtained from Charles River Laboratories, INC (Portage, MI, USA). They were acclimated to the test facility for a minimum of 7 days, and administered the test material seven days/week via oral gavage at dose levels of 0, 5, 25, or 150 mg CS-1246/kg of body weight/day for approximately 10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations. A comprehensive evaluation of male and female reproductive systems was conducted, and included an evaluation of gonadal function, the estrous cycle, mating performance, conception, gestation, parturition and lactation, as well as survival, growth and development of the offspring.

Before administration of test material began, animals were stratified by body weight and then randomly assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard deviations at the start of the study.

The test material was delivered in 4 mL/kg dose volumes, at concentrations in corn oil of 1.25, 6.25, or 37.5 mg/mL. Homogeneity, stability, and analytical verification of the dose levels were determined by GC-MS.

Breeding of the P1 adults commenced after approximately 10 weeks of treatment. Each female was placed with a single male from the same dose level (1:1 mating) until mating occurred or two weeks elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm as an indication of mating. The day on which sperm were detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were separated from the male and returned to their home cage. If a breeding male died or was removed from study, a substitute partner (from the same dose group) that already completed mating was provided. If mating did not occur after two weeks, the animals were separated without further opportunity for mating. Approximately 10 weeks after all F1 litters have been weaned, F1 offspring randomly selected to become P2 adults were bred as described above. Cohabitation of male and female littermates was avoided.

A cage-side examination was conducted at least twice daily. The examination was performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

Clinical examinations were conducted on all males pre-exposure and weekly throughout the study. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14 and 21. Females were observed for signs of parturition beginning on or about GD 20. Females that deliver litters were subsequently evaluated on LD 0, 1, 4, 7, 14, and 21. Clinical observations were not conducted on females that failed to mate or deliver a litter, unless deemed appropriate based on cage-side observations. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

All rats were weighed during the pre-exposure period and weekly during the 10-week pre-breeding periods. Males continued to be weighed weekly after breeding until termination. Mated females were weighed on GD 0, 7, 14, 17, and 21. Lactating females were weighed on LD 1, 4, 7, 14, and 21. Females that failed to mate and/or deliver a litter were weighed weekly during the subsequent gestation and/or lactation segments of the study. Body weight analyses during gestation and lactation were conducted for the following days: GD 0, 7, 14, 21 and LD 1, 4, 7, 14, and 21. Body weight gains of females were calculated for the following intervals in both generations: GD 0-7, 7-14, 14-21 and 0-21 and LD 1-4, 4-7, 7-14 and 14-21 and 1-21. F1 offspring assigned to the P2 generation that began oral gavage dosing on PND 22 were weighed approximately every three days until they reached approximately six weeks of age.

Feed consumption were determined weekly during the 10-week pre-breeding period for all animals by weighing feed containers at the start and end of a measurement cycle. During breeding, feed consumption was not measured due to co-housing. Following breeding, feed consumption for males was measured weekly until termination. For mated females, feed consumption was measured on GD 0, 7, 14, and 21. For females delivering litters, feed consumption was measured on LD 1, 4, 7, 11, 14, 17, 19, and 21. Feed consumption was not measured for females that fail to mate or fail to deliver a litter.

Vaginal lavage samples from all P1 and P2 females were collected daily for three weeks immediately prior to mating and during cohabitation until each female was sperm- or plug-positive or until the two week mating period had elapsed. Lavage samples were collected by irrigating the vagina with water and transferring lavage fluid to a microscope slide to determine estrous cycle length and pattern. On the day of scheduled necropsy, vaginal lavage samples were collected from all P1 and P2 female rats for subsequent determination of the stage of the estrous cycle.

Weights of the left testis and left cauda epididymis were collected for use in calculating sperm count parameters. Sperm parameters were evaluated in all P1 and P2 males at termination. Unless circumstances dictate otherwise, the left and right epididymides and testes were allocated as follows: right epididymis - motility and histopathology; left epididymis - counts; right testis - histopathology; left testis - counts.

Females were observed periodically for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day that one or more delivered fetuses are noted, and was designated as LD 0. The following information will be recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21. Any pup found dead or sacrificed in moribund condition will be sexed and examined grossly, to the extent possible, for external and visceral defects.

To minimize variation in pup growth due to differences in litter size, all litters were standardized to eight pups per litter on PND 4 by randomly ordering the pups in each litter by sex. Pups to be culled were randomly selected using a computer generated randomization procedure, so that four males and four females remained in each litter. If it was not possible to have four pups/sex in each litter, unequal numbers of males and females were retained (e.g., five males, three females). Litters with fewer than eight pups were not culled. Preferential culling of runts was not performed. All litters were weaned on PND 21.

One male and one female per litter were randomly selected as P2 animals to produce the second generation. If there were insufficient litters from which to select P2 animals, additional animals were randomly selected from available litters as needed in order to obtain the required number of animals/dose level. When possible, one pup/sex/litter was randomly selected for a necropsy examination with the collection of organ weights and two additional pups/sex/litter were randomly selected for gross necropsy only. Any weanlings either not held for the next generation adult animals or not selected for necropsy were euthanized by CO2 inhalation and discarded.

All F1 weanlings selected as the future P2 generation were observed daily for vaginal opening beginning on PND 28 or for preputial separation beginning on PND 35. Age and body weight of the animals on the day these markers of puberty onset were acquired were recorded. Examination for puberty onset ceased upon acquisition, or on PND 43 (females) or 53 (males), whichever came first. Animals not acquiring these markers were assigned a value of 43 (females) or 53 (males). If a treatment-related effect on F1 sex ratio or age at pubertal onset was detected, anogenital distance (absolute and relative to the cube root of body weight) was measured in the F2 pups on PND 1.

The necropsy included an examination of the external tissues and all orifices. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The uteri of all females were stained and examined for the presence and number of implantation sites.
Three pups/sex/litter from the F1 and F2 litters randomly selected at the time of weaning were submitted on PND 22 for a complete necropsy. Gross pathological examination was performed as described above for adults, except that the weanlings were not fasted overnight. Representative sample of grossly abnormal tissues and any known target organs were collected from all weanlings at the scheduled necropsy. In addition, one of the three pups/sex/litter were randomly selected from those examined grossly for the collection of brain, spleen, uterus, and thymus weights. Organ-to-body weight ratios were calculated.

Dead or moribund pups were examined in a similar manner for possible defects and/or cause of death.

Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, adrenal glands, spleen, thyroid with parathyroids (weighed after fixation) were recorded, and the organ-to-body weight ratios calculated.

Histologic examination of the tissues were conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrate treatment-related histologic effects at the high dose and relevant gross lesions. Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned , and stained. The presence and integrity of the stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes was examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Examination of the ovaries included enumeration of primordial follicles using a method similar to Bucci et al. (1997). From among the surviving post-lactational P2 females in the control and high-dose groups, 15 per group will be randomly selected for this examination.

Parental body weights, gestation and lactation body weight gains, litter mean body weights, feed consumption, anogenital distance (absolute and relative to the cubed root of body weight), sperm count, follicle count, percent total and progressively motile sperm, mean estrous cycle length and organ weights (absolute and relative) were evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) were performed. If the ANOVA is significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) were performed. Feed consumption values were excluded from analysis if the feed was spilled or scratched.

Gestation length, age at vaginal opening (females), age at preputial separation (males), average time to mating, and litter size were analyzed using a nonparametric ANOVA. If the ANOVA is significant, the Wilcoxon Rank-Sum test with Bonferroni's correction were performed. Sperm morphology were arcsine transformed and analyzed using a parametric ANOVA. Slides containing less than 200 sperm were excluded from analysis. If the ANOVA was significant, the Dunnett's test were performed. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs (1969) and were excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction. Evaluation of the neonatal sex ratio on postnatal day 1 were performed by the binomial distribution test (alpha = 0.05; Steel and Torrie, 1960). Gender was determined for pups born dead on postnatal day 0 and these data were included in sex ratio calculations. Survival indices, post-implantation loss, and other incidence data among neonates were analyzed using the litter as the experimental unit by the censored Wilcoxon test (alpha = 0.05; Hollander and Wolfe, 1973) as modified by Haseman and Hoel (1974) with Bonferroni's correction. Non-pregnant females, females with resorptions only, or females found to be pregnant after staining of their uteri were excluded from gestation and lactation body weights and body weight gains, feed consumption and organ weights. Both the Dunnett's test and Bonferroni's correction corrected for multiple comparisons to the control to keep the experiment-wise error rate at 0.05. Both were reported at the experiment-wise alpha level.

Because numerous measurements were statistically compared in the same group of animals, the overall false positive rate (Type I errors) was greater than the nominal alpha levels. Therefore, the final interpretation of the data considered statistical analyses along with other factors, such as dose-response relationships and whether the results were consistent with other biological and pathological findings and historical control values.

Reproductive indices were calculated for all dose level groups and included female and male mating indices, female and conception indices, female and male fertility index, gestation index, gestation survival index, post-implantation loss, day 1 or 4 pup survival index, and day 7, 14, or 21 pup survival index.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
A cage-side examination was conducted at least twice daily. The examination was performed with the animals in their cages and was designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the animals. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily. Cage-side examinations were also conducted on dams and their litters, at least twice daily. These examinations were conducted as described above.

Clinical examinations were conducted on all males pre-exposure and weekly throughout the study. Clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm-positive or plug-positive) females received clinical examinations on GD 0, 7, 14 and 21. Females were observed for signs of parturition beginning on or about GD 20. Females that deliver litters were subsequently evaluated on LD 0, 1, 4, 7, 14, and 21. Clinical observations were not conducted on females that failed to mate or deliver a litter, unless deemed appropriate based on cage-side observations. Clinical observations included a careful, hand-held examination of the animal with an evaluation of abnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat, and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.
All rats were weighed during the pre-exposure period and weekly during the 10-week pre-breeding periods. Males continued to be weighed weekly after breeding until termination. Mated females were weighed on GD 0, 7, 14, 17, and 21. Lactating females were weighed on LD 1, 4, 7, 14, and 21. Females that failed to mate and/or deliver a litter were weighed weekly during the subsequent gestation and/or lactation segments of the study. Body weight analyses during gestation and lactation were conducted for the following days: GD 0, 7, 14, 21 and LD 1, 4, 7, 14, and 21. Body weight gains of females were calculated for the following intervals in both generations: GD 0-7, 7-14, 14-21 and 0-21 and LD 1-4, 4-7, 7-14 and 14-21 and 1-21. F1 offspring assigned to the P2 generation that began oral gavage dosing on PND 22 were weighed approximately every three days until they reached approximately six weeks of age.
Estrous cyclicity (parental animals):
Vaginal lavage samples from all P1 and P2 females were collected daily for three weeks immediately prior to mating and during cohabitation until each female was sperm- or plug-positive or until the two week mating period had elapsed. Lavage samples were collected by irrigating the vagina with water and transferring lavage fluid to a microscope slide to determine estrous cycle length and pattern. On the day of scheduled necropsy, vaginal lavage samples were collected from all P1 and P2 female rats for subsequent determination of the stage of the estrous cycle.
Sperm parameters (parental animals):
Weights of the left testis and left cauda epididymis were collected for use in calculating sperm count parameters. Sperm parameters were evaluated in all P1 and P2 males at termination. Unless circumstances dictate otherwise, the left and right epididymides and testes were allocated as follows: right epididymis – motility and histopathology; left epididymis – counts; right testis – histopathology; left testis – counts.
Motility
Immediately after euthanasia of males and isolation of their epididymides, a small sample of sperm from the right cauda epididymis was expressed into a dish containing SpermPrep Medium (ZDL, Lexington, Kentucky) and was incubated at room temperature for approximately 2-3 minutes. An aliquot of the incubated sperm suspension was placed in a chamber of the HTM Integrated Visual Optical System (IVOS; Hamilton-Thorne Research, Beverly, Massachusetts) for the determination of total percent motile (showing any motion) and percent progressively motile (showing net forward motion) sperm. Images from the motility analyses were recorded on CD-R and will be archived with the study file. After sperm are released, the epididymis was placed in Bouin’s fixative and subjected to histologic examination.

Counts
The left testis and cauda epididymis were weighed and then frozen at approximately -20°C for subsequent determination of the number of homogenization-resistant spermatids and sperm per testis/cauda epididymis and per gram of testicular/epididymal tissue. The thawed testis or epididymis were minced, diluted and stained with a fluorescent DNA-binding dye (HTM-IDENT, Hamilton-Thorne Research, Beverly, Massachusetts) and the spermatid or sperm count were determined from an aliquot loaded into the IVOS analyzer as described by Stradler et al. (1996). Because there were no treatment-related differences in testicular/epididymal sperm counts, only samples from the high-dose and control animals were evaluated.

Morphology
An aliquot of sperm suspension was placed on a slide, and a smear prepared and air-dried for subsequent evaluation of sperm morphology. At least 200 sperm per male were evaluated and were classified as normal or abnormal as described by Filler (1993). Morphological evaluation of sperm from control and high-dose males was conducted. Sperm morphology were scored blind with respect to treatment group.
Litter observations:
Females were observed periodically for signs of parturition beginning on or about GD 20. In so far as possible, parturition was observed for signs of difficulty or unusual duration. The day of parturition was recorded as the first day that one or more delivered fetuses are noted, and was designated as LD 0. The following information will be recorded for each litter: the date of parturition, the number of live and dead pups on LD 0, 1, 4, 7, 14, and 21, and the sex and body weight of each pup on LD 1, 4 (before and after culling), 7, 14, and 21. Any pup found dead or sacrificed in moribund condition will be sexed and examined grossly, to the extent possible, for external and visceral defects.
To minimize variation in pup growth due to differences in litter size, all litters were standardized to eight pups per litter on PND 4 by randomly ordering the pups in each litter by sex. Pups to be culled were randomly selected using a computer generated randomization procedure, so that four males and four females remained in each litter. If it was not possible to have four pups/sex in each litter, unequal numbers of males and females were retained (e.g., five males, three females). Litters with fewer than eight pups were not culled. Preferential culling of runts was not performed.

All litters were weaned on PND 21. One male and one female per litter were randomly selected as P2 animals to produce the second generation. If there were insufficient litters from which to select P2 animals, additional animals were randomly selected from available litters as needed in order to obtain the required number of animals/dose level. When possible, one pup/sex/litter was randomly selected for a necropsy examination with the collection of organ weights and two additional pups/sex/litter were randomly selected for gross necropsy only. Any weanlings either not held for the next generation adult animals or not selected for necropsy were euthanized by CO2 inhalation and discarded.
All F1 weanlings selected as the future P2 generation were observed daily for vaginal opening beginning on PND 28 or for preputial separation beginning on PND 35. Age and body weight of the animals on the day these markers of puberty onset were acquired were recorded. Examination for puberty onset ceased upon acquisition, or on PND 43 (females) or 53 (males), whichever came first. Animals not acquiring these markers were assigned a value of 43 (females) or 53 (males).
If a treatment-related effect on F1 sex ratio or age at pubertal onset was detected, anogenital distance (absolute and relative to the cube root of body weight) was measured in the F2 pups on PND 1.
Postmortem examinations (parental animals):
Adult males (fasted) were submitted for necropsy sometime after completion of their respective mating period when it was deemed that they were no longer needed for assessment of reproductive effects. Adult females (fasted) were terminated after weaning of their litters, or at least 24 days after the end of the mating period. On the morning of the scheduled necropsy, all surviving P1 and P2 males and females were weighed. The animals were anesthetized by the inhalation of CO2, the tracheas were exposed and clamped, and the animals were euthanized by decapitation. A complete necropsy was conducted on all animals by a veterinary pathologist assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened microscope to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.
The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain for approximately two minutes and were examined for the presence and number of implantation sites.
Representative samples of tissues were collected and preserved in neutral phosphate-buffered 10% formalin, except that the right testis, right epdidymis, and ovaries (P2 females only) were preserved in Bouin’s fixative. Transponders were removed and placed in jars with the tissues.
During routine working hours, any animals found dead or euthanized prior to the scheduled necropsy were necropsied on that day. However, animals euthanized or found dead outside working hours were refrigerated until the next scheduled workday, at which time they were necropsied. Similar necropsy procedures were followed for these animals except that terminal body and organ weights were not recorded and the testes, epididymides and ovaries were preserved in neutral, phosphate-buffered 10% formalin.
Weights of the ovaries, uterus (with oviducts and cervix), testes, epididymides, seminal vesicles with coagulating glands (and fluids), prostate, brain, pituitary (weighed after fixation), liver, kidneys, adrenal glands, spleen, thyroid with parathyroids (weighed after fixation) were recorded, and the organ-to-body weight ratios calculated. In addition, weights of the left testis and left cauda epididymis were collected for use in calculating sperm count parameters.
Weights of the brain, spleen, thymus and uterus from one F1/F2 pups/sex/litter were recorded, and organ-to-body weight ratios were calculated.
Histologic examination of the tissues was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to the liver (P1 and P2 males), stomach (P1 and P2 males and females), relevant gross lesions, and reproductive organs of animals with signs of reduced fertility. In addition, the thyroid glands of the control and high-dose P1 males, and control and high-dose P2 males and females were also examined.

Histopathological examination of the testes included a qualitative assessment of stages of spermatogenesis. A cross section through the approximate center of both testes of control and high-dose males was embedded in paraffin, sectioned, and stained. The presence and integrity of the stages of spermatogenesis was qualitatively evaluated following the criteria and guidance of Russell et al. (1990). Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids, and spermatozoa seen in cross sections of the seminiferous tubules. The progression of these cellular associations defined the cycle of spermatogenesis. In addition, sections of both testes was examined for the presence of degenerative changes (e.g., vacuolation of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis).

Examination of the ovaries included enumeration of primordial follicles using a method similar to Bucci et al. (1997). From among the surviving post-lactational P2 females in the control and high-dose groups, 15 per group were randomly selected for this examination.

Selected histopathologic findings were graded to reflect the severity of the specific lesions.
Postmortem examinations (offspring):
Three pups/sex/litter from the F1 and F2 litters randomly selected at the time of weaning were submitted on PND 22 for a complete necropsy. Gross pathological examination was performed as described above for adults, except that the weanlings were not fasted overnight. Representative sample of grossly abnormal tissues were collected from all weanlings at the scheduled necropsy. In addition, one of the three pups/sex/litter were randomly selected from those examined grossly for the collection of brain, spleen, uterus, and thymus weights. Organ-to-body weight ratios were calculated.
Statistics:
Parental body weights, gestation and lactation body weight gains, litter mean body weights, feed consumption, sperm count, follicle count, percent total and progressively motile sperm, mean estrous cycle length and organ weights (absolute and relative) will be evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or nonparametric (Hollander and Wolfe, 1973) analysis of variance (ANOVA) were performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971) or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Feed consumption values were excluded from analysis if the feed is spilled or scratched.
Reproductive indices:
See below
Offspring viability indices:
See below

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

For deatils, see attachment-1

Effect levels (P0)

open allclose all
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: There were no effects on any parameter of reproductive performance or offspring survival at any dose level tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING): No treatment related effects observed.

CLINICAL SIGNS (OFFSPRING): No treatment related effects observed.

BODY WEIGHT (OFFSPRING): No treatment related effects observed.

SEXUAL MATURATION (OFFSPRING): No treatment related effects observed.

ORGAN WEIGHTS (OFFSPRING): No treatment related effects observed.

GROSS PATHOLOGY (OFFSPRING):There were no treatment-related gross pathologic observations.

HISTOPATHOLOGY (OFFSPRING): There were no treatment-related gross pathologic observations

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
In conclusion, the no-observed-effect level (NOEL) for systemic toxicity was 5 mg/kg/day and the NOEL for reproductive toxicity was 150 mg/kg/day.
Executive summary:

The purpose of this two-generation gavage reproduction toxicity study was to evaluate the potential effects of BIOBAN™ CS-1246 biocide on male and female reproductive function, as well as the survival, growth and development of the offspring. Groups of 27 male and 27 female Crl:CD(SD) rats were administered 0, 5, 25, and 150 mg BIOBAN™ CS-1246 biocide/kg/day for approximately ten weeks prior to breeding, and continuing through breeding, gestation and lactation for two generations. In-life

parameters included clinical observations, feed consumption, body weights, estrous cyclicity, reproductive performance, pup survival, pup body weights, and puberty onset. In addition, post-mortem evaluations included gross pathology, histopathology, organ

weights, oocyte quantitation and sperm count, motility and morphology in adults, and gross pathology and organ weights in weanlings.

A treatment-related decrease in lactation body weight gains over the entire lactation period (lactation days 0-21) was evident in P1 and P2 females given 150 mg/kg/day. Evidence of stomach irritation was seen in the majority of animals given 150 mg/kg/day

and consisted of gross pathological observations of non-glandular mucosal thickening, along with histopathological evidence of hyperplasia of the limiting ridge epithelium, subacute to chronic inflammation of the glandular mucosa and submucosa, and hyperplasia, hypertrophy, and increased mitotic figures in the glandular mucosa.Erosions in the glandular mucosa were also present in some P1 females and some P2 males. Similar effects, albeit of lesser severity and incidence, were seen in animals given 25 mg/kg/day. P1 males given 150 mg/kg/day had increased absolute and relative liver weights accompanied by histopathological findings of slightly altered tinctorial properties (increased eosinophilia) of centrilobular hepatocytes. P1 and P2 males given

150 mg/kg/day had slight treatment-related increases in absolute and relative thyroid gland weights, without associated histopathologic change. There were no other signs of parental toxicity at these or lower dose levels. There were no effects on any parameter of reproductive performance or offspring survival at any dose level tested.

In conclusion, the no-observed-effect level (NOEL) for systemic toxicity was 5 mg/kg/day and the NOEL for reproductive toxicity was 150 mg/kg/day.