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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008, Part B.3, 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: at the beginning of the treatment period, the males were 10 weeks old and the females were 9 weeks old
- Weight at study initiation: at the beginning of the treatment period, the males had a mean body weight of 388 g (range: 355 g to 408 g) and the
females had a mean body weight of 236 g (range: 214 g to 261 g).
- Housing: The animals were individually housed, except during pairing, in wire-mesh cages. A metal tray containing autoclaved sawdust was placed under each cage. Towards the end of the gestation period and during lactation with their litter, the females were individually housed in polycarbonate cages containing autoclaved sawdust. Autoclaved wood shavings were provided as nesting material, a few days before delivery and during the lactation period.
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h / 12 h (7:00 - 19:00)

IN-LIFE DATES: From:16 March 2010 To: 03 June 2010.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
aqueous solution at 0.5%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. The test item was mixed with the required quantity of vehicle in order to achieve the
concentrations of 1, 3 and 5 mg/mL, then homogenized using a magnetic stirrer. The frequency of dosage form preparation was based on available
stability data. The dosage forms were stored at room temperature, protected from light, and delivered to the study room in brown flasks at room
temperature.

VEHICLE
- Concentration in vehicle: 1, 3 and 5 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: each female was placed with the same male until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug and sperm in vaginal smear (referred to as day 0 of post-coitum)
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The HPLC/UV analytical method for the determination of 1,3-Diphenylguanidine in dosage form samples was provided by the Sponsor and this method was validated at CIT prior to dosage form analysis.
The validation was based on the ICH Q2(R1) guideline adopted in October 1994, and accordingly the following parameters were checked:
- specificity,
- precision and accuracy,
- injection repeatability,
- linearity,
- Sensitivity Evaluation Test (SET),
- stability of the test item in working solutions.
The concentration of the test item was determined in samples of each control and test item dosage form prepared for use in weeks 1, 4 and 8. Whenever possible these analyses will be performed prior to administration of the dosage forms to the animals.
Duration of treatment / exposure:
The dosage forms were administered daily according to the following schedule:
. in the males:
- 4 weeks before pairing,
- during the pairing period (2 weeks),
- until sacrifice of the females (at least 10 weeks in total).

. in the females:
- 4 weeks before pairing,
- during the mating period (2 weeks),
- during gestation,
- during lactation until day 4 post-partum inclusive (or until sacrifice),
- until sacrifice for non-pregnant females.

Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
Once daily.
Details on study schedule:
- No F1 parents (only one generation mated).
- Age at mating of the mated animals in the study: approximately 11 weeks for females and 12 weeks for males.
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 15 and 25 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected following the results of a previous 2-week toxicity study in the rat. In this study, dose-levels of 60 and 75 mg/kg/day, when administered to rats of 10 weeks old, caused severe clinical signs of lateral recumbency, loss of balance, staggering gait and convulsions from day 1 or day 2 of dosing, and 66% mortality (first animal prematurely sacrificed on day 1 of dosing). The dose-level of 30 mg/kg/day was well tolerated by the males (only one male showed loss of balance onday 9 of dosing and there were no effects on mean body weight gain), but females showed a greater sensitivity with a 74% lower mean body weight gain than the controls and one female having staggering gait and loss of balance on the second day of dosing.
Positive control:
None.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20
post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food consumed by each male was measured once a week, over a 7-day period, from the first day of treatment until the start of the
pairing period. The quantity of food consumed by each female was measured once a week, over a 7-day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 post-coitum and during lactation for interval days 1-5 post-partum. During the pairing period, the food consumption was measured for neither males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the pairing period, until the
females were mated.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology.
Litter observations:
STANDARDISATION OF LITTERS: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross
anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS: yes, for external and internal abnormalities; possible cause of death was determined for pups born or found
dead.
Postmortem examinations (parental animals):
SACRIFICE:
All surviving F0 males and females and prematurely sacrificed females were deeply anesthetized by an intraperitoneal injection of sodium
pentobarbital and sacrificed by exsanguination.
- males: at final sacrifice of the females (at least 10 weeks of treatment in total),
- females: on day 5 post-partum,
- females which had not delivered: on day 25 post-coitum (after a body weight recording to check for a possible un-noticed delivery),
- mothers with litter dying entirely.

GROSS NECROPSY: on all parent animals including females that were sacrificed prematurely.
- Gross necrospy consisted in examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 5 post-partum.

HISTOPATHOLOGY / ORGAN WEIGHTS:
A microscopic examination was performed on:
- all tissues listed in the tissue procedure table (see section "any other information on material and methods") in the males and females of the control and high dose groups (groups 1 and 4) sacrificed at the end of the treatment period and for females sacrificed prematurely,
- all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment
period,
- all females sacrificed because of no delivery to investigate possible causes.

The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated in all animals sacrificed as scheduled.

Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.
Postmortem examinations (offspring):
Pups found dead and pups sacrificed on day 5 post-partum were carefully examined externally for gross external abnormalities. No tissues were
preserved.

Reproductive indices:
. pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

. post-implantation loss:
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

. mating index:
Number of mated animals
_____________________ x 100
Number of paired animals


. fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs

. gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females

. live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups
Offspring viability indices:
. viability index on day 4 post-partum:
Number of surviving pups on day 4 post-partum
_______________________________________ x 100
Number of live born pups

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Male U25679 (25 mg/kg/day) had staggering gait, lateral decubitus and mydriasis 1 hour after treatment on day 27 of dosing only. These clinical signs were observed in the preliminary study so are known to be related to treatment with the test item in spite of the single occurrence. Female U25754 (25 mg/kg/day) had lateral decubitus, locomotory difficulties and mydriasis on day 1 of lactation only.
Male U25663 (15 mg/kg/day) was aggressive during week 7/8 of treatment and also had soft feces and nodosities on the head during this period.
The etiology of these clinical signs is unknown but given their isolated nature a relationship to treatment with the test item is unlikely. Female U25742 (15 mg/kg/day) had hard abdomen and pallor of eyes from day 1 of lactation. The pups of this female all survived until scheduled sacrifice on day 5 of lactation without clinical signs.
Salivation (recorded as ptyalism) was observed at 15 and 25 mg/kg/day generally from week 6 or week 2 respectively (including during gestation and lactation for some females) and was likely to be related to the taste of the test item and was therefore considered to be non-adverse.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Females U25756 (25 mg/kg/day), U25739 (5 mg/kg/day) and U25721 (0 mg/kg/day) were sacrificed following death of their litters on day 3 or 4 of lactation. No relevant clinical signs, macroscopic or microscopic findings were observed in the females.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated at 25 mg/kg/day had periods of statistically significantly lower mean body weight gain which contributed to an overall mean body weight gain 20% (p<0.05) lower than that of the controls. There were no effects on mean female body weight gains at this dose-level during the pre-mating, gestation or lactation periods.
There were no effects of treatment with the test item on male or female body weight gains at 5 or 15 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean male food consumption was generally comparable with that of the controls throughout the study.
Females treated at 25 mg/kg/day had comparable mean food consumption with the controls during the pre-mating period but had lower mean food consumption during the gestation period, achieving statistical significance from day 14 to day 20 of gestation (29 g/animal/day vs. 35 g/animal/day, p<0.01). Food consumption remained slightly lower during the lactation period.
There were no effects of treatment with the test item on mean female food consumption at 5 or 15 mg/kg/day.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item-related organ weights were recorded.
Statistically significant lower absolute kidney weights were observed in the females given 5 or 25 mg/kg/day. In view of the absence of dose-relationship, the low magnitude of this change and the lack of corresponding microscopic finding, it was considered not to be related to test item.
The other organ weight changes were also not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, were not dose-related in magnitude, and/or were not consistent between the sexes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related macroscopic findings were noted.
The few macroscopic findings noted were those commonly recorded in the Sprague-Dawley rat and none were considered to be related to test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
The examination of liver, kidney and testis/epididymis or ovaries/oviducts from animals of groups 1 and 4 did not show any test item-related microscopic findings.
All microscopic findings noted in treated animals were considered as incidental changes, as they occurred also in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There were no indications of abnormal estrous cycles when compared with those of the control females in any test item-treated female.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item at 25 mg/kg/day or on sperm motility at 5 or 15 mg/kg/day.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no effects of treatment with the test item on mating; all females mated and the highest number of non-mated males was in the control group. The mean number of days of pairing before mating was less than the controls at all dose-levels. In addition, only one female (U25757; 25 mg/kg/day) was not pregnant.
The mean duration of gestation, the mean numbers of corpora lutea, implantations and pups delivered and the mean pre- and post-implantation losses were all comparable with the controls for all dose-levels.
Control female U25724 was pregnant but did not deliver. At macroscopic examination, there was one live fetus and one early resorption in the left uterine horn but no indications of why no delivery occurred.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: lower body weight gain in males at 25 mg/kg/day
Dose descriptor:
NOEL
Remarks:
reproductive performance (mating and fertility)
Effect level:
>= 25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There was an increased incidence of hypoactivity at 15 and 25 mg/kg/day; 12 pups from one litter at 15 mg/kg/day and three pups from one litter at 25 mg/kg/day vs. no control pups. All pups from the group treated at 25 mg/kg/day which were hypoactive were also noted to be cold to the touch. Another pup from the group treated at 25 mg/kg/day had a malrotated forepaw. Given the low number of litters involved, however, it was considered that these clinical signs were unrelated to treatment with the test item.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
The number of mortalities was similar between the control and high-dose groups and lower in the low- and intermediate-dose groups.
It was considered that there were no effects of treatment with the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Both male and female pups from the group treated at 25 mg/kg/day gained less body weight during the lactation period (-29% and -31%, for males and females respectively) and had lower mean body weights on day 5 of lactation (-14% and -15%, for males and females, respectively).
No statistical significance was achieved.
There were no effects at 5 or 15 mg/kg/day.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
The percentage of male pups in the group treated at 25 mg/kg/day was minimally and non statistically significantly low; 39% on day 1 of lactation and 36% of day 5 of lactation. The differences were insufficiently distinct to claim an effect of treatment with the test item.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
15 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lower mean body weight gain over the lactation period and lower mean body weights on lactation day 5 at 25 mg/kg/day

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
1,3-Diphenylguanidine,was administered daily by oral gavage to male and female Sprague Dawley rats, for 4 weeks before mating, during mating, gestation and until day 5 post partum, at dose-levels of 5, 15 or 25 mg/kg/day. Treatment at 25 mg/kg/day resulted in lower mean male body weight gain over the treatment period and lower mean female food consumption during gestation and lactation. One male and one female had lateral decubitus, mydriasis and abnormal locomotion on one occasion. Mean pup body weights on day 5 of lactation and body weight gains over the lactation period were lower, although there were no treatment-related clinical signs and pup mortality was comparable with that of the controls. There were no systemic signs of adverse toxicity at 5 or 15 mg/kg/day. Salivation was observed in several animals treated at 15 mg/kg/day and most of the animals treated at 25 mg/kg/day but this was considered non-adverse. There were no effects on mating, fertility, gestation or delivery at any dose-level. Males treated at 25 mg/kg/day showed no effects on sperm parameters and there were also no effects on sperm motility at 5 or 15 mg/kg/day. There were no effects on organ weights nor any treatment-related macroscopic or microscopic findings.

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be
15 mg/kg/day, the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 25 mg/kg/day and the NOEL for toxic effects on progeny was 15 mg/kg/day.
Executive summary:

The objective of this study was to evaluate the potential toxic effects of 1,3-Diphenylguanidine, following daily oral administration (gavage) to male and female rats from before mating, through mating and gestation and until day 4post-partum. This study provides initial information on possible toxicological effects likely to arise from repeated exposure on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of the conceptus and parturition. The study was conducted as a reproduction/developmental screening test according to OECD 421 guideline and GLP.

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, 1,3-Diphenylguanidine, daily, by oral (gavage) administration, 4 weeks before mating, through mating and gestation and until day 4 post-partum.The dose-levels were 5, 15 or 25 mg/kg/day. Another group of 10 males and 10 females received the vehicle, 0.5% aqueous methylcellulose, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg/day. Clinical signs and mortality were checked once or twice daily, respectively. Body weight and food consumption were recorded weekly. The animals were paired for mating after 4 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs and pup body weights were recorded on days 1 and 5 post-partum. The males were sacrificed after a total of 10 weeks of treatment (after most of the females and litters had been sacrificed). The body and selected organs were weighed and a complete macroscopic post-mortem examination was performed. Sperm samples were taken from the left epididymis and the left testis for assessment of sperm motility, morphology and/or count. A microscopic examination was performed on the reproductive organs, liver and kidneys from the males in the control and high-dose groups and on all macroscopic lesions. The dams were sacrificed on day 5post-partum,the body and selected organs were weighed and a complete macroscopic examination was performed. A microscopic examination was performed on the ovaries, liver and kidneys of the females in the control and high-dose groups and on all macroscopic lesions. Pups were sacrificed on day 5post-partumand, including those found dead, were carefully examined for gross external abnormalities.

 

There were no unscheduled deaths during the study. Lateral decubitus, mydriasis and staggering gait/locomotory difficulties were observed in one male and one female treated at 25 mg/kg/day on one occasion only. Salivation was observed with a dose-related incidence at 15 and 25 mg/kg/day but was considered to be non-adverse. The male group treated at 25 mg/kg/day had a statistically significantly lower mean body weight gain over the treatment period. There were no effects on the females treated at 25 mg/kg/day nor on the groups treated at 5 or 15 mg/kg/day. There were no effects on mean male food consumption. The female group treated at 25 mg/kg/day had lower mean food consumption during gestation and lactation, achieving statistical significance for the period of the last 7 days of gestation. There were no effects on the female groups treated at 5 or 15 mg/kg/day.

There were no effects on mating, fertility, gestation or delivery at any dose-level. Male and female pups from the group treated at 25 mg/kg/day had lower mean body weight gain over the lactation period and lower mean body weights on lactation day 5. Clinical signs were considered to be unrelated to treatment with the test item.

There were no effects of treatment with 1,3-diphenylguanidine on sperm analysis, organ weights, macroscopicpost-mortemexamination and Microscopic examination

 

Based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 15 mg/kg/day, the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 25 mg/kg/day and the NOEL for toxic effects on progeny was 15 mg/kg/day.