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EC number: 203-002-1
CAS number: 102-06-7
CHEMICAL ANALYSIS OF THE DOSE FORMULATIONS
The test item concentrations in the administered dose formulations analyzed in premating, mating, gestation and lactation periods of the P-generation and in postweaning, premating, gestation and lactation periods of the F1 generations remained within an acceptable range of variations (-5.3% to +5.1%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was observed in the control dose formulation.
The objective of this study was to provide general information concerning reproductive and developmental effects of the test item, 1,3-Diphenylguanidine, following daily oral administration (gavage) that may occur as a result of pre- and post-natal exposure as well as an evaluation of systemic toxicity in pregnant and lactating females and young and adult offspring.
The dose formulations were administered to sexually-mature male and female rats (Parental (P) generation) by oral gavage, at test item dose levels of 0, 5, 15 or 25 mg/kg/day, daily, starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, pups were also treated daily by oral gavage, at test item dose levels of 0, 5, 15 or 25 mg/kg/day.
Pups were assigned to Cohorts for reproductive/developmental toxicity testing (Cohorts 1A and 1B, including the production of an F2 generation),
P generation and F1 lactating offspring:
Three groups of 24 male and 24 female Sprague-Dawley rats (P generation) received the test item, 1,3-Diphenylguanidine (batch No. 18051468027), daily for 2 weeks prior to pairing, during pairing, through gestation and lactation until weaning of the F1 pups [Day 21 post-partum (p.p.)]. The test item was administered orally (gavage, 5 mL/kg). A control group of 24 males and 24 females received the vehicle alone (0.5% methylcellulose in drinking water treated by reverse osmosis), under the same experimental conditions, and acted as a reference control group.
The test item was administered orally, by gavage (5 mL/kg) to groups of 20 rats/sex (Cohorts 1A and 1B) receiving 0 (0.5% methylcellulose in drinking water treated by reverse osmosis), 5, 15 or 25 mg/kg/day. The treatment schedules were the following:
Examination of Parental, F1 and/or F2 generations
Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals.
P generation and Cohort 1B males and females were paired until mated or until 14 days had elapsed.
Females from the P generation and Cohort 1B were allowed to deliver normally and rear their progeny. Pregnancy and litter parameters were recorded.
During lactation, the F1 and F2 pups were observed daily for survival and clinical signs. Body weight was measured at designated intervals and the sex-ratio was recorded. In F1, the size of each litter was adjusted on Day 4 p.p. to obtain ten pups per litter. Pup physical and/or reflex development was assessed at designated time-points.
Examination of Cohorts
Cohort 1A: animals were selected for assessment of general toxicity and effects on their reproductive system. Estrous cycle stages were determined daily for all females after the onset of vaginal patency, until the first cornified smear was recorded (estrus), and for 19 days before the end of the treatment period.
Cohort 1B: animals were selected for follow-up assessment of reproductive performance (by mating F1 animals) and to potentially obtain additional histopathology data.
A macroscopic post-mortem examination was performed on all P and F1 animals (including F1 pups culled on Day 4 p.p. and F1 pups not selected on Day 22 p.p.) and F2 pups. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was given to the reproductive organs. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated and organs were weighed wet as soon as possible after dissection. A microscopic examination was performed on all macroscopic lesions and a complete list of tissues from animals in all groups or the control and high-dose groups.
P generation and Cohort 1A: the first 10 surviving animals/sex/group were fasted (food only) for an overnight period of at least 14 hours prior to blood sampling (for hematology, coagulation and blood biochemistry) and urine collection (for urinalysis) at termination. A series of sperm cell evaluations was performed on surviving males from all groups or the control and high-dose groups: motility, morphology, sperm cell head count in testicular/epididymal tissues.
Cohort 1A: splenic lymphocyte subpopulation analysis was performed on 10 surviving animals/sex/group from Cohort 1A.
Thyroid hormone levels (P generation, F1 culled pups, Cohort 1A animals and F2 pups): prior to blood sampling the animals were not fasted (except for animals from which samples were collected for hematology, blood biochemistry and urinalysis). Blood samples were taken on Day 4 p.p. (F1 and F2 pups) for measurements of thyroid hormone (T4) levels, and on Day 22 p.p. (F1 pups not selected for Cohorts) and at termination from the first ten surviving males/group and the first ten lactating females/group (P and Cohort 1A animals), for measurements of thyroid hormone (T4) and Thyroid Stimulating Hormone (TSH) levels.
The test item concentrations in the administered dose formulations analyzed during each period (P-premating, P-mating, P-gestation, P-lactation, F1-postweaning, F1-premating, F1-gestation and F1-lactation) remained within an acceptable range of variations (-5.3% to +5.1%) when compared to the nominal values (± 15% of the nominal concentrations). No test item was detected in the control dose formulation.
Mortality: there were no unscheduled deaths in males. In pregnant females and from 5 mg/kg/day, there were premature deaths considered to be test item related. Namely two females were sacrificed for humane ground at 25 mg/kg/day, two were sacrificed due to the difficulties to deliver (one at 15 mg/kg/day and one at 25 mg/kg/day). In addition, five females (one at 5 mg/kg/day, one at 15 mg/kg/day and three at 25 mg/kg/day) were sacrificed due to the death of their litters.
Clinical signs: in both sexes, there were dose-related increases in ptyalism that were considered to be test item-related, but not adverse as this is a common finding observed after oral gavage. At 25 mg/kg/day and in females at the end of the pregnancy period (around parturition), there was a series of clinical signs suggestive of neurologic disorders (clonic convulsion, locomotory difficulties, loss of balance, staggering gait and/or tonic seizures) which were transient and observed after dosing. These findings were considered to be test-item treatment related and adverse.
Mean body weight, mean body weight change and mean food consumption: there were no adverse effects during the premating, mating, gestation or lactation periods.
Estrous cycle, mating and fertility: there were no adverse effects on estrous cycle, mating (including the mean number of days taken to mate) or fertility.
Gestation: there was a trend towards increased gestation periods in test-item treated females. Two females at 5 mg/kg/day had 24-day gestation periods associated with high pup mortality rates (90-100%). At 25 mg/kg/day, there was an increased number of females with 23-day gestation periods associated with high post-implantation losses (27.1 vs. 15.6% in controls, p<0.05). This finding also observed in Cohort 1B was considered to be test item treatment-related and adverse.
Delivery data: there was a dose-related tendency towards lower number of viable pups from 5 mg/kg/day, despite an absence of statistically significant difference vs. controls or when compared with Historical Control Data. At 25 mg/kg/day, on Day 1 p.p, the number of viable pups was down to 9.3 vs. 12.0 in controls and live birth index down to 72.8 vs. 95.1% in controls. These findings were considered to be test item treatment-related and adverse.
P generation offspring (pre-weaning F1 pups): from 5 mg/kg/day and when compared with controls, there was a statistically significant higher percentage of pups found dead, missing and/or cannibalized on Days 1-4 p.p. (12.5, 16.2 and 32.3%, respectively at 5, 15 and 25 mg/kg/day, vs. 4.9% in controls, with p<0.01 or <0.001). At necropsy of found dead pups, absence of milk and autolysis were noted with high incidence in the treated groups when compared to the control group. Absence of milk in the stomach may represent nursing difficulties or absence of maternal care. These findings were considered to be test item treatment-related and adverse.
Laboratory investigations: there were no test item treatment-related effects on hematology, coagulation, blood biochemistry or urinalysis in P generation animals. In P generation females and at 25 mg/kg/day, when compared with controls or Historical Control Data, there were high mean T4 concentration (+38%, p<0.05) associated with low mean TSH concentrations (-14%). Taking into account the amplitude of the change and the association of the effects, a test item-relationship cannot be excluded but considered to be non-adverse in the absence of finding at pathology examination. There were no effects at 15 and 5 mg/kg/day.
Sperm analysis: there were no effects on sperm analysis parameters (motility, morphology, sperm/testicular numerations or daily sperm production rate).
Mortality: there were no unscheduled deaths.
Clinical signs: ptyalism was observed in all groups (including controls) but with an increased incidence in test item-treated groups when compared with controls. This finding is commonly observed after gavage and was considered to be test item treatment-related but not adverse.
Body weight, body weight change and food consumption: there were no adverse effects.
Sexual development: there were no effects.
Estrous cycles: there were no effects on mean time to first estrous after vaginal opening and on mean estrous cycle parameters.
Laboratory investigations: there were no adverse test item treatment-related effects on hematology, coagulation, blood biochemistry or urinalysis in P generation animals. There were no effects on mean thyroid hormones levels (T4 and TSH).
Lymphocyte subtyping (Cohort 1A): there were no test item treatment-related findings.
Mortality: there were no unscheduled deaths in males. In pregnant females and from 15 mg/kg/day, there were premature deaths considered to be test item related. Namely two females were sacrificed for humane ground at 25 mg/kg/day, one was sacrificed due to the difficulties to deliver at 25 mg/kg/day and four were sacrificed due to the death of their litters (two at 15 mg/kg/day and two at 25 mg/kg/day)..
Clinical signs: in both sexes, there were dose-related increases in ptyalism that were considered to be test item-related, but not adverse as this is a common finding observed after oral gavage. At 25 mg/kg/day and in both sexes, there was a series of clinical signs suggestive of neurologic disorders (e.g. clonic/tonic convulsion, loss of balance and/or staggering gait) which were observed after dosing and considered to be test item-related and adverse.
Body weight, body weight change and food consumption: there were no adverse effects on mean body weight, mean body weight change or mean food consumption.
Estrous cycles, mating and fertility: there were no effects.
Gestation: at 25 mg/kg/day, mean duration of gestation was increased (22.3 vs. 21.9 days in controls, p<0.05). This finding was considered to be test-item related (already observed in P generation) and adverse. There were dose-related increased post-implantation losses from 5 mg/kg/day.
Delivery: as a consequence of increased post-implantation losses, there were lower live birth indexes at 15 and 25 mg/kg/day (84.7% and 67.5% vs. 100.0% in controls, respectively) and lower number of live pups at 25 mg/kg/day (7.8 vs. 11.4 in controls, p<0.01). These findings were considered to be test-item related and adverse.
F1 generation offspring (pre-weaning F2 pups): from 15 mg/kg/day and when compared with controls, there were low viability indexes on Day 4 p.p. (down to 56.6% at 25 mg/kg/day vs. 99.0% in controls or 93.1 % in Historical Control Data). At external examination, this finding was associated with pups cold to the touch from 5 mg/kg/day and/or with emaciated appearance at 25 mg/kg/day. These findings were considered to be test-item related and adverse. They were considered to represent lack of maternal care as confirmed with the increased number of found dead pups with autolysis and/or absence of milk in the stomach. There were no deaths after Day 4 p.p. in pre-weaning F2 pups.
P generation: There was a dose-related increased incidence of mortality in test item-treated females at ≥ 15 mg/kg/day due to reproductive trouble, and at 25 mg/kg/day with not evident cause of death. Increased liver weights were recorded in males treated at ≥ 15 mg/kg/day and correlated with the microscopic hepatocellular hypertrophy. There were no gross test item-related findings. Dose-related minimal non-adverse hepatocellular hypertrophy was noted in the liver in males and females treated at 25 mg/kg/day. At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the high-dose and the control groups.
Cohort 1A: There was no mortality. Increased liver weights were recorded in females treated at ≥ 5 mg/kg/day and in males treated at 25 mg/kg/day. This correlated with hepatocellular hypertrophy at microscopic examination. There were no gross test item-related findings. Dose-related minimal hepatocellular hypertrophy was noted in the liver from in males and females treated at 25 mg/kg/day. At quantitative evaluation of primordium follicles or corpora lutea, there were no differences between the test item-treated groups and the controls.
Cohort 1B: Test item-related mortality was noted at 15 and 25 mg/kg/day. Reproduction trouble (dead litter or difficulties to deliver) were noted for two females treated at 15 mg/kg/day and for three females treated at 25 mg/kg/day. No evident cause could be established for two females treated at 25 mg/kg/day. Increased liver weights were recorded in males and females treated at ≥ 15 mg/kg/day. No microscopic findings were considered to be test item-related (examination of brain and macroscopic findings).
Non-selected pups: There was no mortality. There were not test item-related organ weight differences. There were no test item-related gross changes.
The test item, 1.3-Diphenylguanidine, was administered daily by oral gavage, at dose levels of 0, 5, 15 or 25 mg/kg/day, to sexually-mature male and female rats (parental (P) generation) starting 2 weeks before mating and continuously through mating, gestation and weaning of their pups (F1 generation). At weaning, the F1 generation was also exposed to graduated doses of the test item and was assigned to Cohorts of animals for reproductive/developmental toxicity.
Systemic toxicity evaluation:
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 15 mg/kg/day based on the severe clinical signs suggestive of neurologic disorders in males and females, leading to premature euthanasia in females for humane grounds at 25 mg/kg/day.
Reproductive/developmental toxicity evaluation:
The No Observed Adverse Effect Level (NOAEL) for reproductive/developmental toxicity was considered to be lower than 5 mg/kg/day based on the increased gestation periods at all dose-levels, reproductive troubles in females (found dead litters from 5 mg/kg/day, difficulties to deliver from 15 mg/kg/day) resulting in high incidence of pup mortality at birth and on the first days of lactation from 5 mg/kg/day, not associated with maternal neurotoxicity which was only observed at the top dose-level of 25 mg/kg/day.
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