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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental dates: 11/17/2006 to 8/3/2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline, GLP

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
Qualifier:
according to
Guideline:
other: EEC No. 0378-6978
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Test material: Biocide CS1135This breaks down to produce formaldehyde and 2-amino-2-methylpropanol

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Male and female Sprague-Dawley rats (27/sex/dose) were obtained from Charles River Laboratories INC (Portage, MI, USA), and were 6 weeks of age at study initiation. Rats were acclimated to the testing facitility prior to random assignment to dose groups, and were offered feed and water ad libitum. They were housed in rooms designed to maintain adequate environmental conditions for the species (air changes, photocycle, temperature, and relative humidity).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
Groups of 27 male and 27 female Crl:CD(SD) rats were administered the test material seven days/week via oral gavage at dose levels of 0, 20, 60, or 200 mg CS-1135/kg of body weight/day (in polyethylene glycol 400 vehicle) 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.
Details on mating procedure:
Breeding of the P1 adults commencee 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 had 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 was detected or a vaginal copulatory plug was observed in situ was considered GD 0. The sperm- or plug-positive (presumed pregnant) females were then 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 has already completed mating was provided, if available. If mating had not occurred after two weeks, the animals were separated without further opportunity for mating. Approximately 10 weeks after all F1 litters were weaned, F1 offspring were randomly selected to become P2 adults and were bred as described above. Cohabitation of male and female littermates was avoided.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose confirmation of CS-1135 in polyethylene glycol -400 (PEG) analyzed using gas chromatography with electron impact mass spectrometry detection (GC/EI/MSD) and quantified using isotopically labeled internal standard. Dose confirmation within +/- 14% of nominal concentrations
Duration of treatment / exposure:
10 weeks prior to breeding and continuing through breeding (two weeks), gestation (three weeks) and lactation (three weeks) for each of two generations. F1 pups were exposed indirectly through milk and by gavage beginning at weaning.
Frequency of treatment:
once daily, 7-days/week
Doses / concentrations
Remarks:
Doses / Concentrations:0, 20, 60, 200 mk/kg/dayBasis:nominal conc.
No. of animals per sex per dose:
27/sex/group
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
Cage-side examinations were conducted at least twice daily. This examination was typically 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. Any animals found dead were necropsied as soon as practical. Cage-side examinations were be conducted on dams and their litters, 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. Clinical observations were not conducted on females that failed to mate or deliver a litter, unless deemed appropriate based on cageside 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 were 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. F1 offspring assigned to the P2 generation that begin oral gavage dosing on PND 22 were weighed approximately every three days until they reached six weeks of age. The additional body weights collected were used for dose calculations to accommodate for rapid growth during this time.Feed consumption was 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 continued to be 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 failed to mate or failed to deliver a litter.
Oestrous cyclicity (parental animals):
Vaginal lavage samples from all P1 and P2 females were collected daily for three weeks 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 gently irrigating the vagina with water and transferring lavage fluid to a microscope slide. Vaginal lavage slides collected during the three weeks prior to mating were examined microscopically 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):
Sperm parameters were evaluated in all P1 and P2 males at termination. Unless circumstances dictated 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.MotilityImmediately 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 were archived with the study file. After sperm were released, the epididymis was placed in Bouin's fixative and subjected to histologic examination.CountsThe 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 determined from an aliquot loaded into the IVOS analyzer as described by Stradler et al. (1996). Initially, samples from the high-dose and control animals were evaluated. If treatment-related effects were seen, the middle and low-dose groups will also be evaluated as necessary to establish a NOEL.MorphologyAn 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 classified as normal or abnormal as described by Filler (1993). Morphological evaluation of sperm from control and high-dose males was conducted. If treatment-related effects were observed, evaluation of sperm from the lower dose levels was performed. Sperm morphology was 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 was 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 was sexed and examined grossly, to the extent possible, for external and visceral defects. Cage-side examinations were be conducted on litters, at least twice daily.To minimize variation in pup growth due to differences in litter size, all litters were standardized to eight pups per litter on PND 4. This was accomplished 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. Culled pups were euthanized and then discarded. All litters were weaned on PND 21. All F1 weanlings selected for mating, were observed daily for vaginal opening beginning on PND 28 (Cooper et al., 1989) or preputial separation beginning on day 35 (Korenbrot et al., 1977). The age and body weight at the time of landmark acquisition were recorded. Examination for puberty onset ceased upon acquisition, or on PND 43 (females) or 62 (males), whichever came first.
Postmortem examinations (parental animals):
A complete necropsy was conducted on all adult animals. The necropsy included an examination of the external tissues and all orifices. 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) and known target organs will be 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. Histologic examination of the tissues (Table A6.8.2/01-1) was conducted on all control and high-dose adult rats. Examination of tissues from the remaining groups was limited to those tissues that demonstrated treatment-related histologic effects at the high dose, and relevant gross lesions and reproductive organs of animals with signs of reduced fertility. 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 at 5 µm 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 defines the cycle of spermatogenesis. In addition, sections of both testes were 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 wiere graded to reflect the severity of specific lesions
Postmortem examinations (offspring):
Three pups/sex/litter from the F1 and F2 litters randomly selected at the time of weaning will be 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.
Statistics:
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) 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) will be 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) will be performed. Feed consumption values will be excluded from analysis if the feed is spilled or scratched.Gestation length, age at vaginal opening (females), age at preputial separation (males), average time to mating, and litter size will be analyzed using a nonparametric ANOVA. If the ANOVA is significant, the Wilcoxon Rank-Sum test with Bonferroni's correction will be performed. Sperm morphology will be arcsine transformed and analyzed using a parametric ANOVA. Slides containing less than 200 sperm will be excluded from analysis. If the ANOVA is significant, the Dunnett's test will be performed. Statistical outliers (alpha = 0.02) will be identified by the sequential method of Grubbs (1969) and will only be excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices will be analyzed by the Fisher exact probability test
Reproductive indices:
Reproductive indices were calculated for all dose level groups as follows:Female mating index = (No. females with evidence of mating/No. paired) x 100Male mating index = (No. males with evidence of mating/No. paired) x 100Female conception index = (No. females with evidence of pregnancy/No. mated) x 100Male conception index = (No. males siring a litter/No. mated) x 100Female fertility index = (No. females with evidence of pregnancy/No. paired) x 100Male fertility index = (No. males siring a litter/No. paired) x 100Gestation index = (No. females delivering a viable litter/No. females with evidence of pregnancy) x 100Gestation survival index = percentage of delivered pups alive at birthPost-implantation loss = (No. implants – No. viable offspring)/(No. implants) x 100
Offspring viability indices:
The following information was 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.Day 1 or 4 pup survival index = (No. viable pups on day 1 or 4/No. born live) x 100Day 7, 14, or 21 pup survival index = (No. viable pups on day 7, 14 or 21/No. live after culling) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Gastric irritation due to release of formaldehyde
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)Treatment-related clinical observations in the P1 animals included one high-dose male that was euthanized in moribund condition. Necropsy revealed marked stomach ulceration and inflammation in the nasal region as contributors to the moribund condition of this rat. Gastric irritation due to release of formaldehyde from the test material likely caused this treatment-related effect.TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)Analyses of dose solutions for concentration verification were conducted on four different occasions during the study, representing all dose levels, sexes and major study phases. The mean concentrations of CS-1135 in the vehicle for all analyses ranged from 85.8 to 105.9% of the targeted concentrations. The overall mean concentration for the entire study ranged from 92.3 to 95.9% of the targeted concentrations. Analyses also confirmed that the test material was homogeneously distributed in the vehicle with relative standard deviations ranging from 0.5 to 4.7%.ORGAN WEIGHTS (PARENTAL ANIMALS)The absolute and relative liver weights of P1 males and females given 200 mg/kg/day were heavier than the controls, statistically identified, outside of the historical control range and were interpreted to be treatment-related. These higher liver weights were associated with microscopic altered staining of hepatocytes and vacuolization consistent with fatty change in males and females given 200 mg/kg/day. The absolute and relative liver weights of P1 males given 60 mg/kg/day were outside of the historical control data, but were not statistically identified. These higher liver weights were accompanied by treatment-related microscopic liver effects and were interpreted to be treatment-related. The liver weights of females given 20 or 60 mg/kg/day were not affected by treatment. The absolute and relative kidney weights of P1 males given 200 mg/kg/day were higher than the controls. Although only the increase in absolute kidney weight was statistically identified, both the absolute and relative kidney weights were outside of the historical control range and were interpreted to be treatment-related. These higher kidney weights were not associated with any microscopic treatment-related effects in the kidneys and may reflect an adaptive change associated with the excretion of the test material in the urine.GROSS PATHOLOGY (PARENTAL ANIMALS)The only organ affected by treatment was the nonglandular stomach. The nonglandular stomach was thickened in the majority of males and females given 200 mg/kg/day. In addition, a few females in this group also had an erosion or ulcer in the glandular mucosa. These treatment-related effects were confined to rats in the 200 mg/kg/day group and were suggestive of an adaptive response to irritation associated with the presence of formaldehyde as a degradation product of the test material. As was observed in the P1 adults, the only organ affected by treatment in the P2 adults was the stomach. The nonglandular stomach was thickened in the majority of the males and females given 200 mg/kg/day. Two males and one female in this dose group also had erosions or ulcers in glandular mucosa of the stomach.HISTOPATHOLOGY (PARENTAL ANIMALS)The primary treatment-related effect was observed in the stomachs of males and females given 200 mg/kg/day, and to a lesser extent in females given 60 mg/kg/day. These stomach effects were likely due to point of contact irritation, associated with the presence of formaldehyde as a degradation product of the test material. In the nonglandular stomach the effects were primarily characterized by hyperkeratosis and hyperplasia of the mucosa. In females the nonglandular effects were more focal and primarily affected the limiting ridge of the stomach. In addition, this minimal effect in the limiting ridge also affected several more rats in the 60 mg/kg/day rats than was observed in the low dose or control groups. This portion of the stomach is normally lined by squamous epithelium and this effect was interpreted to be an adaptive response to an irritant material. This was the region of the stomach with the grossly recognizable changes noted during necropsy. The glandular stomachs of P1 males and females given 200 mg/kg/day were also significantly affected in a number of animals and interpreted to be reflective of contact with an irritant material. Hyperplasia of the glandular mucosa of the stomach was evident in the majority of P1 males and females given 200 mg/kg/day, with the effect being slightly more severe in females than males. In addition, some of the rats given 200 mg/kg/day had one or more mucosal erosions and ulcers with an associated inflammation and edema in the mucosa or submucosa of the glandular region of the stomach. In the 60 mg/kg/day group, a single female also had a mucosal erosion with edema in the submucosa of the glandular stomach. This rat also had acute, focal, glandular inflammation in the mucosa. Several more females given 60 mg/kg/day had subacute to chronic inflammation in the glandular submucosa, that was either focal or multifocal, and very slight in degree, when compared to the incidences in the control or 20 mg/kg/day group. The erosions, ulcers, edema, hyperplasia and inflammatory infiltrates primarily involved the fundic portion of the glandular stomach. The microscopic findings in the liver of males and females given 60 or 200 mg/kg/day were considered treatment-related. The microscopic findings were characterized by 1) altered tinctorial properties; increased eosinophilia; hepatocyte; centrilobular of a very slight or slight degree; and 2) vacuolization consistent with fatty change, hepatocyte; individual cells; multifocal, very slight. All of the other microscopic findings in the remaining tissues were spontaneous occurrences or associated with a gavage incident. The primary treatment-related effect was observed in the stomachs of P2 males and females at 200 mg/kg/day. As in the P1 generation, these stomach effects were likely due to point of contact irritation, associated with the presence of formaldehyde as a degradation product of the test material. In contrast to the minimal effect observed in the P1 60 mg/kg/day female stomach, this dose group was not affected in the P2 group. The responses in nonglandular and glandular portions of the stomach were identical to those in the P1 males and females with only slight numerical differences. The microscopic findings in the liver which appeared to be treatment-related were the same in P2 males and females as in P1 rats. Vacuolization and altered tinctorial properties of the hepatocytes were the same. The changes in both were apparent in males at 60 and 200 mg/kg/day. Several more males had a slight grade of vacuolization in these groups in contrast to a very slight grade in P1 males. In the P1 females there were fewer control and 20 mg/kg/day rats with vacuolization of hepatocytes in contrast to the P2 groups. Therefore, the liver in P2 60 mg/kg/day females was unaffected. In the male P2 livers there was a slight increase in the number of rats with multifocal, and slight degree of aggregates of macrophages as histocytes. The increased kidney weights and adrenal weights were not associated with any microscopic findings due to the test chemical. All of the other microscopic findings observed in other tissues were considered normal spontaneous events or associated with changes secondary to gavage accidents. Histologic examination of the reproductive organs of animals with signs of reduced fertility did not reveal any effect of treatmentThere were no treatment-related or statistically-identified differences in the mean number of small and growing ovarian follicles in females given 200 mg/kg/day as compared to the control females.OTHER FINDINGS (PARENTAL ANIMALS)There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, time to mating, gestation length, or pup sex ratio in either generation.There was a statistically identified increase in percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) in the mid- and high-dose group of the P1 litter, and the high-dose group of the P2 litter. The increase in postimplantation loss was considered treatment-related for the P1 and P2 high-dose litters because of the statistical significance and because the values were outside of the historical control ranges. The statistically identified increase in postimplantation loss of the mid-dose P1 litter was considered to be a spurious finding unrelated to treatment because the value was within the historical control range, was not present in the P2 litter at this dose level, and was largely driven by one dam with clear signs of maternal toxicity.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
Systemic Toxicity
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive Toxicity
Effect level:
60 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Due to slighlty increased post-implantation loss at 200 mg/kg/day

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight increases in males at treatment levels 60 and 200 mg/kg/day
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Vacuolization and altered tinctorial properties of the heptaocytes in males at 60 and 200 mg/kg/day. Similar results were seen in P1 females at the highest dose level. Effects similar to those seen in stomachs of P0 males and females were seen at the highest dose level
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
not examined

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
Systemis toxicity
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
Vacuolization and altered tinctorial of hepatocytes
Key result
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Due to slightly increased post-implantation losses at 200 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed

Details on results (F1)

VIABILITY (OFFSPRING)There were no effects of treatment at any dose level on mating, conception, fertility or gestation indices, time to mating, gestation length, or pup sex ratio in either generation. In the P1 generation, there was a slight, but not-statistically identified, decrease in gestation survival index in the 200 and 60 mg/kg/day dose groups (96.0 and 95.5%, respectively) compared to controls (99.7%). Animals in the 200 mg/kg/day dose group also had slight, but not-statistically identified, decreases in PND 1 and 4 survival indices (96.5 and 94.8%) compared to controls (99.5 and 98.7). Because these values were not statistically significant, were near or just slightly outside the historical control range, and were not reproduced in the second generation, they were deemed spurious and unrelated to treatment.There was a statistically identified increase in percent postimplantation loss (percentage of live born pups relative to total uterine implantation sites) in the mid- and high-dose group of the P1 litter, and the high-dose group of the P2 litter. The increase in postimplantation loss was considered treatment-related for the P1 and P2 high-dose litters because of the statistical significance and because the values were outside of the historical control ranges. The statistically identified increase in postimplantation loss of the mid-dose P1 litter was considered to be a spurious finding unrelated to treatment because the value was within the historical control range, was not present in the P2 litter at this dose level, and was largely driven by one dam (6158, see Clinical Observations section) with clear signs of maternal toxicity.ORGAN WEIGHTS (OFFSPRING)There were no treatment-related alterations in organ weights of F1 or F2 weanlings at any dose level. See "Observations: parental animals" for results on organ weghts of adult P2 animalsGROSS PATHOLOGY (OFFSPRING)There were no treatment-related gross pathologic observations. See "Observations: parental animals" for results on gross pathology of adult P2 animalsHISTOPATHOLOGY (OFFSPRING)See "Observations: parental animals" for results on gross pathology of adult P2 animals.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Results: F2 generation

Details on results (F2)

There were no effects on any parameter of reporductive performance or offspring growth and survival at 20 or 60 mg/kg/day

Effect levels (F2)

Key result
Dose descriptor:
NOEL
Remarks:
Developmental toxicity
Generation:
F2
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
200 mg/kg bw/day
Treatment related:
yes
Relation to other toxic effects:
not specified
Dose response relationship:
not specified

Applicant's summary and conclusion