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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
multi-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

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: MAFF Japan Test Guidelines for Agricultural Chemicals 2-1-17 Notification 12-Nousan-8147
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Purity: 91.7%

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat was selected for this study because it is a preferred species for reproductive toxicity testing and recommended by regulatory guidelines. The Crl:CD(SD) strain was chosen because extensive background data are available from the literature. This strain is also considered suitable relative to longevity, hardiness, and incidence of spontaneous disease.
Sex:
male/female
Details on test animals and environmental conditions:
Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
Age and Body weight of animals: The P1 generation animals were approximately 54 days old at the start of treatment, and in the body weight ranges of 207.6-272.7 grams (males) and 161.5-218.3 grams (females).
Acclimation period: 7 days
Diet: PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002, ad libitum
Water: Tap water, ad libitum
Housing: All male animals were housed individually during non-mating periods in solid-bottom caging with bedding containing Nestlets™as enrichment. Each cage rack contained only animals of one sex, except during cohabitation when the animals were housed as breeding pairs (female in male’s cage). Fourteen days after the first day of cohabitation, females without evidence of copulation were housed singly. During lactation periods, adult females were housed with their litters.

Environmental conditions:
Temperature: 20-26ºC (68-79ºF)
Humidity: 30-70%
Photoperiod: Alternating 12-hour light and dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose with 0.1% Tween 80
Details on exposure:
Dose volume: 5 mL/kg body weight
Details on mating procedure:
Start of Cohabitation
Animals were co-housed for mating after approximately 10 weeks of exposure to the test substance. The day animals were first co-housed was designated as day 1 of pairing.

Duration of Cohabitation Period
Each female was continually housed on a 1:1 basis with a randomly selected, non-sibling male of the same dietary concentration level, in the male's cage. Animals were co-housed until evidence of copulation was observed or until 2 weeks elapsed. On the day copulation was confirmed, the female was transferred back to individual cage housing. The cohabitation period ended on the morning of day 15 of pairing.

Evidence of Copulation
Once daily, each female was examined for the presence of an intravaginal copulation plug or sperm in the vaginal lavage sample, either of which was considered evidence of copulation. The presence of an intravaginal plug and/or sperm was recorded. The day evidence of copulation was observed was designated as GD 0.

Estrous Cycle Evaluation
Vaginal lavage samples were collected daily from all surviving adult P1 and F1 female rats in order to determine the stage of the estrous cycle. Vaginal lavage samples were collected beginning 3 weeks prior to the start of cohabitation and continuing until copulation was confirmed or the cohabitation period had ended. The vaginal lavage sample collected on the day copulation was confirmed was not used for estrous cycle evaluation.

Gestation Procedures
Beginning on GD 20 for mated females or beginning 7 days after the end of the cohabitation period for females without evidence of copulation, female animals were observed at least twice daily for signs of delivery and offspring.

Lactation Procedures
The day when delivery was complete was designated LD 0 for dams and PND 0 for pups. At each examination period (days 0, 4, 7, 14, and 21 postpartum), offspring were individually handled and examined for abnormal behavior and appearance; any dead or abnormal pups were recorded. Dams that had no live pups remaining during the lactation period were sacrificed at the discretion of the study director; litters of dams that died during lactation were sacrificed.
Individual litter data was recorded on days 0, 4, 7, 14, and 21 postpartum. Where possible, all live and dead pups were sexed. Live pups were individually weighed.
On day 4 postpartum, litters were culled randomly to 8 (4/sex when possible) and the number of pups of each sex recorded. Extra offspring were euthanized (by decapitation) and discarded without anatomic pathology examination. Litters of 8 offspring or fewer were not reduced.
On day 21 postpartum, offspring in the F1 litters of each treatment level were randomly selected (one animal/sex/litter when possible) to be evaluated for sexual maturation and collection of developmental landmark data.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were collected from the top, middle, and bottom of each concentration of the initial dose preparation and were analyzed to verify homogeneity and concentration (average of homogeneity samples) in the formulations.
Samples were also taken from at least 2 more different time points for each of the concentrations.
An additional set of samples was analyzed for homogeneity and concentration due to a change in formulation volume.
Stability of the test sample in the vehicle had been established in a previously conducted study and therefore, was not conducted during this study.
At the time of analysis, the samples were diluted with an appropriate solvent and the diluted samples were analyzed by ultra high-performance liquid chromatography (UHPLC) with ultraviolet (UV) detection.
The analyses results show that the test substance was homogeneously mixed and on target for all concentrations levels analyzed. Stability of test substance in the vehicle in the concentration range from 1 to 200 mg/mL stored at room temperature for up to 15 days.
The test substance was not detected in the control samples.
Duration of treatment / exposure:
Animals were dosed daily by oral gavage for approximately 70 days prior to cohabitation and during the cohabitation period (up to 2 weeks).
P1 and F1 females showing evidence of copulation were dosed throughout gestation.
P1 and F1 females were dosed after delivering litters, until day 21 postpartum.
P1 and F1 females that did not deliver a litter continued to be dosed until the day before sacrifice.
F1 males and females were dosed beginning on day 21 postpartum until the day before scheduled euthanasia.
Frequency of treatment:
Daily
Details on study schedule:
Refer table 1 and 2 (in any other information on materials and methods section)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Dose / conc.:
80 mg/kg bw/day
Dose / conc.:
320 mg/kg bw/day
No. of animals per sex per dose:
30
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 5, 20, 80, and 320 mg/kg/day of the test substance were selected based on results from a one generation reproductive toxicity study in rats and a 90-day oral gavage subchronic toxicity study in rats and in consultation with the sponsor.

Assignement to Groups
1. P1 Generation
Animals of each sex were selected for use on study based on adequate body weight gain and freedom from any clinical signs of disease or injury. They were distributed by computerized, stratified randomization into study groups as designated in the Study Design.
The body weights of the animals were checked on test day 1 to ensure the mean body weights of the test groups were not statistically significantly different from control.
Animals that were not assigned to a test group were released for other laboratory purposes or sacrificed by carbon dioxide asphyxiation while under isoflurane anesthesia and discarded without anatomic pathology evaluation.
2. F1 Generation
On PND 21, offspring in the F1 litters of each treatment level were selected (one animal/sex/litter, when possible) to serve as parents for the F2 generation. For groups without sufficient litters and at the discretion of the study director, additional pups were chosen from randomly selected litters within the group to achieve the required group size. Selection of animals within litters was random.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily to detect moribund or dead animals.
At least once daily and at least 2 hours post-dosing to detect acute clinical signsof systemic toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once in a week

BODY WEIGHT: Yes
- Time schedule for examinations: Once in a week and at sacrifice
P1 and F1 females were weighed on days 0, 7, 14, and 20 during the gestation period and days 0, 7, 14, and 21 during the lactation period.

Food Consumption and Food Efficiency
Individual food consumption was determined weekly for all P1 and F1 animals throughout the period, except during cohabitation, ending at test day 71.
Individual food consumption of presumed pregnant P1 and F1 females was recorded on gestation days (GD) 0, 7, 14, and 20 and on lactation days (LD) 0, 7, and 14. Food consumption was not measured for females without evidence of copulation or for males.

Developmental Landmarks - F1 Generation
1. Vaginal Patency
F1 female animals designated for mating were examined for vaginal patency once daily beginning on PND 21 until achievement or PND 43, whichever came first. Body weight was recorded on the day of achievement.
2. Preputial Separation
F1 male animals designated for mating were examined for preputial separation beginning on PND 35 until achievement or PND 55, whichever came first. Body weight was recorded on the day of achievement.
3. Anogenital Distance
Anogenital distance was not recorded in F2 pups on day 0 postpartum since treatment-related effects were not noted on sex ratio and/or sexual maturation of F1 pups.
Estrous cyclicity (parental animals):
Vaginal lavage samples were collected daily from all surviving adult P1 and F1 female rats in order to determine the stage of the estrous cycle. Vaginal lavage samples were collected beginning 3 weeks prior to the start of cohabitation and continuing until copulation was confirmed or the cohabitation period had ended. The vaginal lavage sample collected on the day copulation was confirmed was not used for estrous cycle evaluation. Vaginal lavage samples were examined microscopically for determination of the stage of the estrous cycle (diestrus, estrus, proestrus).
Sperm parameters (parental animals):
Sperm parameters for all surviving P1 and F1 adult males were evaluated. The right epididymis was removed, and the right cauda epididymis was weighed. Sperm was collected from the right cauda epididymis for evaluation of motility and morphology. The left epididymis and testis were frozen in liquid nitrogen and stored between -65°C and -85°C for sperm and spermatid counts, respectively.
Litter observations:
Litter examinations (live, dead, or missing pups, individual pup weights, clinical observations) were determined at birth, on PND 4, and weekly during the 21-day lactation period.
Postmortem examinations (parental animals):
SACRIFICE: All adult females (and adult males euthanized prior to the scheduled sacrifice) were sacrificed by isoflurane anesthesia and exsanguination. Adult males that survived to the schedule sacrifice were euthanized by CO2 inhalation and exsanguination.

GROSS NECROPSY: At necropsy, P1 and F1 adult rats received a gross pathology examination that included examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. The uteri of all cohabited P1 and F1 adult females were examined for the presence and number of implantation sites.

HISTOPATHOLOGY / ORGAN WEIGHTS: Individual organ weights were determined for all P1 and F1 adults at the scheduled sacrifice. Paired organs were weighed together. Group mean values and organ weight ratios (% body weight and % brain weight) were calculated. Terminal body weights determined just prior to necropsy were used for the calculation of organ/body weight ratios. Male accessory sex gland weights were calculated by additing the weights of the prostate and seminal vesicle with coagulating gland (including fluids).
The tissues were collected from all P1 and F1 adults and preserved in 10% neutral buffered formalin (except for the testis and epididymis which were preserved in modified Davidson’s solution). Kidney and liver were identified as target tissues and were evaluated microscopically from for all parental animals in all groups. The following tissues were evaluated microscopically in the first 10 control and high dose (320 mg/kg/day) P1 and F1 parental rats surviving to scheduled sacrifice: testis, epididymis, prostate, seminal vesicles, coagulating glands, ovary, oviducts, uterus, vagina, and cervix. In addition, the reproductive organs from rats with suspected impaired reproductive performance (i.e., reproductive failures: failed to produce a litter) were evaluated from all groups.
Most gross lesions in P1 and F1 adults were saved and evaluated microscopically. Selected gross observations for which a microscopic diagnosis would not be additive (e.g., osteoarthritis and urinary bladder calculus) were saved, but not processed for microscopic evaluation.

Other: A quantitative evaluation of primordial and growing follicles was conducted on 10 lactating F1 females (surviving to scheduled sacrifice) from control and high-dose (320 mg/kg/day) groups. To clarify findings in the initial evaluation, the remaining treatment groups (10 lactating F1 females per group) were also examined. For each animal evaluated, six ovarian cross sections (5 μm thick) were taken from the central area of the ovary using a step section technique. Primordial and growing follicles (up to but not including antral follicles) were enumerated for up to 12 ovarian sections per animal.
Postmortem examinations (offspring):
SACRIFICE: Pups (nursing offspring) up to lactation day 4 were euthanized by decapitation. For lactating pups on study between lactation days 5 and 20, euthanasia was by an intraperitoneal injection of a commercial euthanasia agent.
On day 21 postpartum, offspring in the F1 and F2 litters that were selected for organ weights and/or postmortem examination were euthanized by isoflurane anesthesia and exsanguination. All remaining weanlings were euthanized by isoflurane followed by CO2 inhalation.

Pups that died during the lactation period or were sacrificed due to the death of the dam underwent a gross pathological evaluation and the carcass was discarded. Organs were not weighed and tissues were not saved.

HISTOPATHOLOGY / ORGAN WEIGHTS: For one weanling/sex/litter, the liver, kidneys, brain, spleen, and thymus were weighed and placed in formalin. Gross lesions were also collected and placed in formalin. Liver and kidney from weanlings in the 0 and 320 mg/kg/day groups were evaluated microscopically.
Statistics:
Separate analyses were performed on the parental data collected for each sex. For litter parameters, the proportion of affected fetuses per litter or the litter mean were used as the experimental unit for statistical evaluation. Significance was judged at p < 0.05.
Refer table 3 (in any other information on materials and methods section) for the Method of Statistical Analysis

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations. The observations that were noted were unremarkable, present in only one or 2 animals, and not dose dependent.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
In P1 males, there were no test substance-related unscheduled deaths. Two males, one (animal #218) from the 5 mg/kg/day and one (animal #312) from the 20 mg/kg/day were found dead prior to scheduled euthanasia. The deaths that were noted were unremarkable, present in only one or 2 animals, and not dose dependent, thus were considered spurious. In P1 females, there were no early deaths on this study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In P1 males, there were statistically significant reductions in mean body weight at 80 and 320 mg/kg/day that were evident beginning after 2 weeks of test substance administration that generally persisted throughout the rest of the study. The magnitude of the reductions was 8%/10% or lower at 80 and 320 mg/kg/day, respectively compared to controls. There were no test substance-related effects on mean body weight at 20 mg/kg/day or lower.
There were statistically significant, treatment-related effects on body weight gain at 80 and 320 mg/kg/day. The magnitude of these statistically significant reductions ranged from 14-19% and 12-23% lower than control at 80 and 320 mg/kg/day, respectively. As a result of the reduced body weight gains observed throughout the study, the mean overall (TD 1-120) body weight gains were 11% and 14% lower than control at 80 and 320 mg/kg/day, respectively.
There were no test substance-related effects on mean body weight gains at 20 mg/kg/day or lower. Occasional instances of statistically significant decreases observerd at 5 or 20 mg/kg/day were not considered test substance-related or adverse because they were minimal in magnitude and/or not dose dependent, or had no impact on overall body weight gains. Cumulative mean body weight gain was within 5% of the control group mean at all dietary concentrations tested.
There were no treatment-related effects on body weight or weight gain in P1 females throughout premating, gestation, and lactation at any level tested. The single instance of statistically significantly increased body weight gain observed at 80 mg/kg/day was considered to be spurious and not treatment-related since the body weight gains were increased compared to control, were not dose dependent, and had no impact on overall body weight gains.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In P1 males, there were no test substance-related reductions in mean food consumption at any level tested. Mean overall (TD 1-71) food consumption was within 6% of the control group at 320 mg/kg/day. There were, however, treatment-related, statistically significant effects on mean food efficiency observed at 80 and 320 mg/kg/day in P1 males. Cumulative mean food efficiency was 9% and 12% lower than control values at 80 and 320 mg/kg/day, respectively. These reductions in food efficiency were considered treatment-related and potentially adverse.
There were no treatment-related effects on food consumption parameters in P1 females throughout premating, gestation, and lactation at any level tested. The single instance of statistically significantly increased food efficiency observed at 80 mg/kg/day was considered to be spurious and not treatment-related since the food effieicency was increased compared to control, was not dose dependent, and had no impact on overall mean food efficiency.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related, statistically significant effects on mean food efficiency observed at 80 and 320 mg/kg/day in P1 males. Cumulative mean food efficiency was 9% and 12% lower than control values at 80 and 320 mg/kg/day, respectively. These reductions in food efficiency were considered treatment-related and potentially adverse. There were no treatment-related effects on food consumption parameters in P1 females throughout premating, gestation, and lactation at any level tested. The single instance of statistically significantly increased food efficiency observed at 80 mg/kg/day was considered to be spurious and not treatment-related since the food effieicency was increased compared to control, was not dose dependent, and had no impact on overall mean food efficiency.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test substance-related organ weight changes were present in the liver and/or kidney of P1 males and females. Kidney weight parameters were increased in P1 males and females. Minimal (≤ 10% above control) test substance-related increases in liver weight parameters were present in P1 males at 80 and 320 mg/kg/day and in P1 females at 320 mg/kg/day.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Adverse, test substance-related degeneration of renal tubules in the outer stripe of the outer medulla of the kidney was present in P1 males and females at 320 mg/kg/day. Renal tubular degeneration was associated with dilatation of tubules in the in the 320 mg/kg/day male P1 groups. Minimal test substance-related centrilobular hypertrophy was present in the liver of P1 males and females at 320 mg/kg/day, and in the P1 females at 80 mg/kg/day.
Minimal test substance-related centrilobular hypertrophy was present in the liver of males and females of both the P1 and F1 generations at 320 mg/kg/day, and in the P1 females at 80 mg/kg/day. Hypertrophy was characterized by an increase in size and cytoplasmic esosinophilic staining of hepatocytes surrounding central veins. A slight increase in yellow-brown pigment within Kuppfer cells was occcassionally present in some areas of centrilobular hypertrophy. Hepatocellular hypertrophy was associated with minimal increases in liver weight at the 320 mg/kg/day dose level but was not asscoated with microscopic changes indicative of hepatocellular injury. Therefore, centrilobular hypertrophy was considered to represent a non-adverse adaptive response associated with metabolism of the test compound.
An increased incidence of minimal clear cell/mixed foci was present in 8/30 P1 males in the 320 mg/kg/day group. Foci generally occurred as single, small circumscribed areas of hepatocytes with clear cytoplasm or a mixture of hepatocytes with clear and pale deosinophilic cytoplasm. As noted above for liver hypertrophy, foci were not associated with evidence of hepatocellular injury. Therefore, this finding was considered to be possibly test substance related but non-adverse. Low incidences (one or two/group) of clear cell/mixed foci were seen in other treated groups. However, foci in these groups did not occur in a clear dose-related manner and were not considered to be test substance related.

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related effects on estrous cycle parameters in P1 or F1 females at any level tested. The single instance of statistical significance observed in mean number of cycles in P1 females at 320 mg/kg/day was not considered treatment-related or adverse since there were no changes in mean cycle length at any level tested. Additionally, there were no effects observed in estrous cycle parameters in F1 females at any level tested. These F1 animals have potentially been exposed to the test substance for a longer duration and during critical phases of postnatal development. Therefore, this instance of statistical significance for mean number of estrous cycles at 320 mg/kg/day in P1 females was not considered treatment-related or adverse.
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
320 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Remarks on result:
other: No effects at highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: based on body weight parameter effects in P1 males at ≥80 mg/kg/day and adverse effects observed in the kidneys of P1 and F1 adult animals

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related clinical observations during any phase of the study. The observations that were noted were unremarkable, present in single animals, and not dose dependent.
There were no test substance-related clinical observations in F1 pups. All observations were considered unremarkable, were infrequently reported, and were not dose dependent.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
In F1 males, there were no test substance-related unscheduled deaths. One male (animal #4006) from the 80 mg/kg/day and one male animal #5001) from the 320 mg/kg/day group died prior to scheduled euthanasia. The deaths that were noted were unremarkable, present in only one or 2 animals, and not dose dependent, thus were considered spurious.
For the F1 offspring, there were no test-substance related effects on litter sizes, sex ratio, or on survival throughout lactation (live born index, viability index, lactation index). The single instance of statistical significance was considered to be spurious since the change was not dose dependent.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 Males
There were treatment-related, statistically significant effects on body weight and body weight gain in F1 males at 320 mg/kg/day. At weaning (TD 1), mean body weights were generally comparable to control group at all levels tested. However, after approximately 4 weeks of direct dosing of the test substance to animals at 320 mg/kg/day, mean body weights were reduced 6-9% lower than control group throughout the rest of the study. These changes were statistically significantly different from control.
Mean body weight gains were generally lower than control throughout the study at 320 mg/kg/day. Mean overall (TD 1-106) body weight gains were 9% (statistically significant) lower than control group at this level. The magnitidue of change for these F1 males was not as significant as was observed for for P1 males at this level, compared to concurrent controls. However, these changes in F1 males at 320 mg/kg/day were still considered potentially adverse and treatment-related.
F1 Females
There were no treatment-related effects on body weight or weight gain in F1 females throughout premating, gestation, and lactation at any level tested. Occasional instances of statistically significant effects observed at 20 or 320 mg/kg/day were not considered test substance-related or adverse because they were minimal in magnitude and/or not dose dependent, or had no impact on overall body weight gains.
There were no treatment-related effects on mean pup weights at any level tested for either P1 or F1 generation litters. Mean pup weights were within 4% of control group means at all levels tested for both generations.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 Males
In F1 males, there were no test substance-related reductions in mean food consumption parameters at any level tested. Mean overall (TD 1-71) food consumption was within 4% of the control group at 320 mg/kg/day. Occasional instances of statistically significant effects observed were not considered test substance-related or adverse because they were either minimal in magnitude and/or not dose dependent, or had no impact on overall food parameters.
F1 Females
In F1 females, there were no test substance-related reductions in mean food consumption parameters at any level tested. Occasional instances of statistically significant effects observed at 20 mg/kg/day or 320 mg/kg/day were not considered adverse because they had no impact on overall food consumption parameters and/or they were not dose dependent.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Kidney weight parameters were increased in F1 males and females administered 80 or 320 mg/kg/day. Kidney weight changes were associated with adverse microscopic changes in the kidney in all but the 80 mg/kg/day F1 female group. In the F1 parental generation, liver weight increase was limited to the 320 mg/kg/day female group.
In the F1 parental generation, liver weight increase were limited to the 320 mg/kg/day female group.However, there were no microscopic changes in the liver indicative of liver injury. Therefore, the increases in liver weight were considered to be non-adverse adaptive changes associated with metabolism of the test substance.
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
Degeneration of renal tubules in the outer stripe of the outer medulla of the kidney was present in rats at ≥80 mg/kg/day. It occurred in F1 males and females at 320 mg/kg/day and in all but the F1 females at 80 mg/kg/day. These changes were dose-related with respect to both incidence and severity and were considered test substance related and adverse. Tubular degeneration was more severe in males than female.
Minimal test substance-related centrilobular hypertrophy was present in the liver of F1 males and females at 320 mg/kg/day. Hypertrophy was characterized by an increase in size and cytoplasmic esosinophilic staining of hepatocytes surrounding central veins. A slight increase in yellow-brown pigment within Kuppfer cells was occcassionally present in some areas of centrilobular hypertrophy. Hepatocellular hypertrophy was associated with minimal increases in liver weight at the 320 mg/kg/day dose level but was not asscoated with microscopic changes indicative of hepatocellular injury. Therefore, centrilobular hypertrophy was considered to represent a non-adverse adaptive response associated with metabolism of the test compound.
Description (incidence and severity):
Ovarian Follicle Counts: There were no treatment-related differences in the total number of primordial and pre-antral follicles in F1 adult females at any concentration tested. A statistically higher group mean follic le count was present in the 20 mg/kg/day group. However, this increase did not occur in a dose-related manner and was likely due to 4/10 individual ovarian follicle counts from the control group that were be low the laboratory’s historical control range (58-223 counts). Therefore, this difference was not considered to be test substance-related.

F1 Animals - Developmental Landmarks: In F1 males and females, there were no test substance-related effects on developmental landmark acheivment (vaginal patency or preputial separation) at any level tested. Values for mean days to achievement were comparable across all dietary concentrations tested.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
320 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reproductive performance
Key result
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
20 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on body weight parameter effects in P1 males at ≥80 mg/kg/day and adverse effects obser ved in the kidneys of P1 and F1 adult animals

Results: F2 generation

General toxicity (F2)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test substance-related clinical observations in F2 pups. All observations were considered unremarkable, were infrequently reported, and were not dose dependent.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
For F2 offspring, there were no test-substance related effects on litter sizes, sex ratio, or on survival throughout lactation (live born index, viability index, lactation index). The single instance of statistical significance was considered to be spurious since the change was not dose dependent.
Body weight and weight changes:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test substance-related organ weight changes in F2 weanlings.
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, non-treatment-related

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
320 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: no adverse test-related effects observed at the highest dose

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
There was no test substance-related mortality nor were there any test substance-related clinical observations. Test substance-related, potentially adverse effects included statistically significant effects on body weight parameters in P1 males at ≥80 mg/kg/day and F1 males at 320 mg/kg/day and reduced food efficiency in P1 males at 320 mg/kg/day. There were no treatment-related effects observed on any in-life parameter at 20 mg/kg/day or lower.
Adverse, test substance-related degeneration of renal tubules in the outer stripe of the outer medulla of the kidney was present in males and females of both the P1 and F1 adult generations at 320 mg/kg/day and in all 80 mg/kg/day adult rats except the F1 females. These changes were generally more severe in males than females, and in the P1 compared to F1 generation. Renal tubular degeneration was associated with dilatation of tubules in the 320 mg/kg/day male P1 and F1 groups. Kidney weights were also increased in the 80 and 320 mg/kg/day groups. Minimal test substance-related centrilobular hypertrophy was present in the liver of males and females of both the P1 and F1 generations at 320 mg/kg/say, and in the P1 females at 80 mg/kg/day. Hepatocellular hypertrophy was associated with minimal increases in liver weight at the 320 mg/kg/day dose level, but was not associated with microscopic changes indicative of hepatocellular injury. Therefore, centrilobular hypertrophy and increased liver weights were considered to be non-adverse adaptive responses associated with metabolism of the test substance. Similarly, clear cell foci, which were increased in incidence in the liver of P1 males at 320 mg/kg/day, were minimal, focal, and not associated with degenerative changes in the liver, and were therefore considered to be non-adverse.
There was no evidence of adverse reproductive toxicity or adverse effects on offspring at any concentration tested for either the P1 or F1 generations. The data for mating, fertility, precoital interval length, gestation length, and implantation site counts were comparable across all groups tested for each respective generation. Additionally, there were no adverse, test substance-related effects noted on pup survival indices, estrous parameters, or sperm parameters at any concentration for either generation.
Therefore, the No-Adverse-Effect-Level (NOAEL) was 20 mg/kg/day for systemic toxicity based on body weight parameter effects in P1 males at ≥80 mg/kg/day and adverse effects observed in the kidneys of P1 and F1 adult animals. The NOAEL for reproductive toxicity was 320 mg/kg/day, the highest level tested.
Executive summary:

The objective of this study was to evaluate the effect of the test substance on the gonadal function, conception, parturition, and growth and development of male and female Crl:CD(SD) rats over 2 generations involving the production of one set of litters in each generation. The test substance was administered orally because it is a potential route for human exposure and is a route recommended by regulatory agencies for this type of study. In the current study, groups of Crl:CD(SD) rats (30/sex/concentration) were exposed to the test substance via oral gavage at dose levels of 0, 5, 20, 80, and 320 mg/kg/day. Samples of the formulations were analyzed and confirmed to be at targeted concentrations and homogeneously mixed under the conditions of use for the study. Stability of test substance in the vehicle in the concentration range from 1 to 200 mg/mL stored at room temperature for up to 15 days had been established in a previous study.

Following at least 10 weeks of exposure to the test substance (premating), the P1and F1 generation males and females were co-housed within their respective treatment groups to produce F1and F2 litters, respectively. Dams were allowed to deliver and rear their offspring until weaning on postnatal day 21 (PND 21). F1and F2 litters were culled to 4 pups/sex/litter (litter size permitting) on PND 4; all remaining pups were discarded without further evaluation. At weaning, selected F1offspring (one rat per sex per litter when possible) were randomly selected to serve as parents for the F2 generation. F2 litters were terminated at weaning.

Clinical observations, body weight, and food consumption were determined weekly throughout the study. Litter examinations (live, dead, or missing pups, individual pup weights, clinical observations) were determined at birth, on PND 4, and weekly during the 21-day lactation period. Estrous cycle parameters were evaluated daily for 3 weeks prior to cohabitation and up to the day of presumed mating in P1 and F1 adult rats. The age at either vaginal opening or preputial separation was recorded for the F1 generation. Sperm motility, morphology, concentration in the cauda epididymis, and spermatid concentration in the testis were determined for P1 and F1 adult rats at the terminal sacrifice.

Gross postmortem examinations were performed on selected animals, and selected organs were weighed and/or retained for histopathological examination. A quantitative evaluation of ovarian follicles was conducted on selected F1 females.

Under the current experimental conditions during the in-life period, there was no test substance-related mortality nor were there any test substance-related clinical observations. Test substance-related, potentially adverse effects included statistically significant effects on body weight parameters in P1 males at ≥80 mg/kg/day and F1 males at 320 mg/kg/day and reduced food efficiency in P1 males at 320 mg/kg/day. There were no treatment-related effects observed on any in-life parameter at 20 mg/kg/day or lower.

Adverse, test substance-related degeneration of renal tubules in the outer stripe of the outer medulla of the kidney was present in males and females of both the P1 and F1 adult generations at 320 mg/kg/day and in all 80 mg/kg/day animals except the F1 females. These changes were generally more severe in males than females, and in the P1 compared to F1 generation. Renal tubular degeneration was associated with dilatation of tubules in the in the 320 mg/kg/day male P1 and F1 groups. Kidney weights were also increased in the 80 and 320 mg/kg/day groups. Minimal test substance-related centrilobular hypertrophy was present in the liver of males and females of both the P1 and F1 generations at 320 mg/kg/day, and in the P1females at 80 mg/kg/day. Hepatocellular hypertrophy was associated with minimal increases in liver weight at the 320 mg/kg/day dose level, but was not associated with microscopic changes indicative of hepatocellular injury. Therefore, centrilobular hypertrophy and increased liver weights were considered to be non-adverse adaptive responses associated with metabolism of the test substance. Similarly, clear cell foci, which were increased in incidence in the liver of P1 males at 320 mg/kg/day, were minimal, focal, and not associated with degenerative changes in the liver, and were therefore considered to be non-adverse.

There was no evidence of adverse reproductive toxicity or adverse effects on offspring at any concentration tested for either the P1 or F1 generations. The data for mating, fertility, precoital interval length, gestation length, and implantation site counts were comparable across all groups tested for each respective generation. Additionally, there were no adverse, test substance-related effects noted on pup survival indices, estrous parameters, or sperm parameters at any concentration for either generation.

Therefore, the No-Adverse-Effect-Level (NOAEL) was 20 mg/kg/day for systemic toxicity based on body weight parameter effects in P1 males at ≥80 mg/kg/day and adverse effects observed in the kidneys of P1 and F1 adult animals. The NOAEL for reproductive toxicity was 320 mg/kg/day, the highest level tested.