Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Taken into account the reliable studies, 1,3-diphenylguanidine did not directlyaffect the fertility of male mice and male and female rats when administered by gavage up to the maximal tested dose level of 16 and 25 mg/kg/d, respectively. Very conservative NOAELs, based on the effects on the reproductive organs, secondary to malnutrition and exhaustion, can be established at 32 mg/kg bw/d for rats.
No adverse effect on reproductive organs or fertility were observed in the recent screening study (Davies 2010) at the highest dose of 25 mg/kg/day.

Link to relevant study records
Reference
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
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
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.
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.
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.
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
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
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: 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.
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
Critical effects observed:
no
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
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
Critical effects observed:
no
Reproductive effects observed:
not specified
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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
32 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Davis study is a reliable study with a klimisch score of 1.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Effects on fertility

 

The potential toxic effects of 1,3-Diphenylguanidine (purity 98.9%) was evaluated, following daily oral administration (gavage) to male and female rats from before mating, through mating and gestation and until day 4 post-partum (Davis, 2010). 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 5post-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 macroscopicpost-mortemexamination 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 5 post-partum and, 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.

 

In an attent to repeat the reprotoxic effects reported in the Bempong’ studies (ultimately considered to be not reliable K3 (OECD, 2002), 1,3-Diphenylguanidine (purity 99.9%) was administered by daily gavage to groups of 25 male CD-1 mice at dose levels of 0, 0.06, 0.25, 1, 4 and 16 mg/kg/d during an 8-week pre-mating period (Van Marwijk and Koëter, 1989; Koëter et al., 1992). Females were not dosed at any time during the study. Within 24 hours after the last treatment, 9 to 13 males, randomly taken from each group were killed and subject to gross examination at autopsy. A selected number of organs were weighted and preserved. Sperm abnormality evaluation was performed in the selected males from the control and 16 mg/kg dose group. The remaining males in the control, 4 and 16 mg/kg body wt per day groups were mated with non-dosed females. Reproductive performance, necropsy findings and litter data were recorded.

No differences were found between control and dosed groups in body weight gain during the dosing period, macroscopic observations and organ weights at necropsy. Microscopic examination of the testes in the 16 mg/kg/d group, did not show any effect due to 1,3-diphenylguanidine dosing when compared to the control group. Sperm abnormality evaluation in the 16 mg/kg/d group showed a slight but statistically significant increase (5% versus 2% in control) in sperm with folded tails but normal heads. However, since the total number of abnormal sperm cells as well as the number of specified sperm abnormalities was similar, the observed increased number of sperm cells with folded tails is considered of doubtful significance. Male and female fertility as well as reproduction performance were comparable in the groups examined (0, 4 and 16 mg/kg/d). Maternal necropsy findings and litter data did not reveal any dose-related effect.

It was concluded that under the conditions of this study, 1,3-diphenylguanidine did not exert any significant adverse effects on fertility, reproductive capacity or embryonic/foetal development in CD-1 mice when administered to males at levels up to 16 mg/kg/d (Koëter et al., 1992; WTR, 1989; reliability 2).

 

In addition to the results of the sub-chronic and sub-acute toxicity studies on the rat and mouse described in section “Repeated dose toxicity” (Irwin, 1995; Murata et al., 2000), special studies for recognising reproductive toxic effects were also performed

 

Male and female F344/N rats and B6C3F1 mice had been administered feed having a 1,3-diphenylguanidine (purity 98.9%) content of 250, 500, 750, 1,500 and 3,000 ppm for 13 weeks (Irwin, 1995). At the end of the 13-week studies, vaginal cytology and sperm motility evaluations were performed on all rats in the 0, 500, 750, and 1500 ppm groups and in all mice in the 0, 250, 750 and 3000 ppm groups.

Female rats exhibited uterine hypoplasia and a prolonged reproductive cycle in the 750 ppm-group (ca. 49 mg/kg b.w. and day) and 1,500 ppm-group (ca. 95 mg/kg b.w. and day) in comparison with the controls. All females of the 3,000 ppm-group died during the study, so that comparable studies of these animals could not take place. The male rats, only in the 1,500 ppm-group (ca. 100 mg/kg b.w. and day), showed diminished sperm motility. Alterations in the reproductive organs (e.g. depletion of the prostate, hypospermia, reduced spermatogenesis) were occasionally found in the males of the 3,000 ppm-group (ca. 181 mg/kg b.w. and day). Also for the mice which obtained up to 3,000 ppm 1,3-diphenylguanidine in feed for 13 weeks, a prolonged reproductive cycle was observed in the females of the highest concentration group and, in the male animals, a decreased sperm motility. The increased number of spermatid heads in the testes paired with the lower number of sperm in the epididymis is evidence against an effect on spermatogenesis, whereas an effect on the release of sperm into the epididymis cannot be excluded.

Comparisons of the parameter changes determined after the 1,3-diphenylguanidine-feeding in the rat and mouse, which can be used for assessing a possible reproductive toxic effect, with the results of tests with feed withdrawal (Chapin et al., 1993a and 1993b; Levin et al., 1993) infer that the effects observed in the 1,3-diphenylguanidine-treated animals in high concentration groups are a result of the poor general state of health (malnutrition, exhaustion) of the animals.


Effects on developmental toxicity

Description of key information
In female rats and mice foetotoxic, but not teratogenic, effects were seen after the oral administration of maternotoxic doses. In the rat study the NOAEL was given as 25 mg/kg bw for the foetuses. In the mouse study the NOAEL was given as higher than 10 mg/kg bw for the foetuses.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines 560/6-82-001
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Michigan)
- Age at study initiation: 91 days approx.
- Weight at study initiation: 220 g approx.
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): ad libitum (Purina certified rodent chow #5002)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 20 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72°C +/-3°F
- Humidity (%): 40% or above
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hr light / 12 hr dark
Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A specified amount of the test article DPG was weighed into a glass weigh boat for each group, transferred to a mortar and ground with the vehicle until a slurry was obtained. The slurry was transferred to a volumetric flask via a series of vehicle rinses. Vehicle was then added in sufficient quantity to achieve the appropriate concentration for each dose group. The flasks were inverted several times and stirred for 5 to 10 minutes. The mixtures were then transferred to amber dosage jars. The test mixtures were prepared fresh daily.
A dosage volume of 10 ml/kg was used for all dosage levels.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose (methocel)
- Lot/batch no. (if required): 820-7112-A
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
The animals were paired for mating in the home cage if the male.
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 M /1 F
- Length of cohabitation: until prove of pregnancy
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6-15 of gestation
Frequency of treatment:
once daily
Duration of test:
15 days
Remarks:
Doses / Concentrations:
5, 25 or 50 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females / dose
Control animals:
yes, concurrent vehicle
Details on study design:
No
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, each morning
DETAILED CLINICAL OBSERVATIONS: Yes, daily during all the gestation
BODY WEIGHT: Yes , on gestation days 0, 6, 9, 12, 16 and 20
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Each fetus was individually weighed, sexed and tagged for identification.
- External examinations: Yes: all per litter (eyes, palate, external orifices)
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No data
Statistics:
All analyses were conducted using two-tailed tests for a minimum significance level of 5% comparing the treatment group to the control group; all means have been presented with standard deviations. The number of animals (N) used to calculate the means has been provided on the individual data tables. All statistical tests were performed by a Digital Computer with appropriate programming as referenced below.
1. The fetal sex ratios were compared by the Chi-square test with Yates’ correction factor.
2. The numbers of litters with malformations and variations were compared by Fisher's Exact Test.
3. The numbers of early and late resorptions, dead fetuses and post-implantation losses were compared by the Mann-Whitney U-test.
4. Mean numbers of corpora lutea, total implantations, viable fetuses, mean fetal and maternal body weight at each interval and maternal body weight
gains were analyzed by a one-way analysis of variance, and Dunnett's test.
Indices:
No
Historical control data:
No
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Alopecia on the forepaws and forelegs was observed in one a nimal in the 50 mg/kg/day group prior to dosing on gestation day 6 and in all study groups during the treatment period, with an increased incidence and duration noted in the 50 mg/kg/day group. During the treatment period, hair loss was extensive in the 50 mg/kg/day group in the pelvic, abdominal, thoracic, urogenital, inguinal, dorsal back and tail areas. All animals in this dose group were lethargic and had tachypnea and decreased limbtone during the treatment period and with one exception all animals were prostrate and ataxic. A few animals were hypersensitive to the touch, salivated and had piloerection during the treatment period. Clonic convulsions, lacrimation, clear nasal discharge, dried red material around the nose, red urogenital discharge and yellow urogenital matting were observed as single incidences in the 50 mg/kg/day group. Lethargic behavior, salivation prior to dosing, hair loss in the pelvic and abdominal areas and dried brown material around the mouth were each noted once in different animals in the 25 mg/kg/day group and may be related to treatment with DPG. No clinical signs of toxicity were observed in the 5 mg/kg/day group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean maternal body weight gain in the 50 mg/kg/day dose group was significantly decreased at all intervals during the treatment period. The most severe decrease (p < 0.01) occurred during the last four days of treatment (gestation days 12-16). The mean body weight gain in the 50 mg/kg/day group was very slightly increased after the treatment period (gestation days 16-20) when compared to the vehicle control group. This resulted in significantly decreased (p < 0.01) body weight gains for the entire gestation period (days 0-20). Group mean body weights were slightly decreased on gestation day 9 and significantly decreased at p < 0.01 on gestation days 12, 16 and 20 in the 50 mg/kg/day group. Mean body weight gain in the 25 mg/kg/day group was very slightly decreased during the overall treatment period (gestation days 6-16) when compared to the vehicle control group. This effect may be related to treatment as there was also a very slight increase in body weight gain following treatment. However, mean body weights in the 25 mg/kg/day group were comparable to the vehicle control group throughout gestation. Body weights and body weight gains in the 5 mg/kg/day group were not affected by treatment with DPG.
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on maternal toxic effects:
In all groups treated with DPG, foetal sex ratios, the mean numbers of viable foetuses, implantation sites and corpora lutea were comparable to the vehicle control group. Mean foetal weights in the 5 and 25 mg/kg/day groups were comparable to the vehicle control. Mean foetal weight in the 50 mg/kg/day group was significantly reduced (p < 0.05) when compared to the vehicle control group. Mean postimplantation loss was slightly increased in the 5 mg/kg/day group due to one female with twelve early resorptions. This increase was not considered biologically meaningful since the effect was not observed at the 25 mg/kg/day dose level. An increase in mean post-implantation loss was also apparent in the 50 mg/kg/day group. One female in the 50 mg/kg/day had all five of the late resorptions occurring in this study, which may be a secondary effect of maternal toxicity. Internal gross necropsy findings for females sacrificed at the scheduled laparotomy such as cystic ovaries, pitted kidneys, white foci or nodules on the lungs and hydronephrosis are considered normal for animals of this strain and age and could not be attributed to the compound.
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Abnormalities:
not specified
Details on embryotoxic / teratogenic effects:
The infrequent occurrence of foetal malformations observed in this study was not indicative of a response to treatment with DPG; each study group, including the control, had one foetus with malformations. One foetus in the control group had multiple anomalies including vertebral agenesis, mandibular microagnathia, a dome-shaped head and microphthalmia. Situs inversus was observed in one foetus in the 5 mg/kg/day group, anophthalmia and internal hydrocephaly were observed in one foetus in the 25 mg/kg/day group and a thread-like tail with anal atresia was observed in one foetus in the 50 mg/kg/day group. Developmental variations observed in the DPG groups were similar to those in the control group except for an increase in the number of fetuses with unossified sternebrae (#5 and/or #6), reduced ossification of the thirteenth ribs, 25 presacral vertebrae and bent ribs in the 50 mg/kg/day group. Reduced ossification would be expected in view of the foetal body weight inhibition at this dose level. The increased number of foetuses with bent ribs in the 50 mg/kg/day dose group is probably associated with maternal toxicity. Although three foetuses from one dam in the 25 mg/kg/day group had bent ribs, the incidence is within the range of our historical control data. In addition, maternal toxicity was slight at this dose level and foetal body weight inhibition was not apparent. The expression of bent ribs at the 25 mg/kg/day dose level was not considered compound-related.
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
> 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In conclusion, DPG induced severe maternal toxicity at a dose level of 50 mg/kg/day. Fetotoxicity was also expressed at this dose level by a significantly reduced mean fetal body weight and by an increase in fetal variations. A dose level of 25 mg/kg/day was considered a marginal "no-effect" level for maternal toxicity; a fetotoxic response was not apparent. A dose level of 5 mg/kg/day was considered a "no-effect" level.
Executive summary:

Potential maternal, embryotoxic and teratogenic effects of DPG were evaluated in this study in rats. DPG was admixed in 0.5% aqueous Methocel and administered orally by gavage to three groups of 25 bred Charles River COBS CD female rats as a single daily dose from days 6 through 15 of gestation. Dose levels of5, 25 and 50 mg/kg/day were selected. For comparative purposes, 25 control females were concurrently dosed with 0.5% aqueous Methocelon a comparable regimen at 10ml/kg/day. Throughout gestation, all females were observed twice daily for toxicity and body weights were recorded at appropriate intervals. On day 20 of gestation, all surviving females were sacrificed for Cesarean section; fetuses were weighed, sexed and examined for external, skeletal and soft tissue anomalies and developmental variations.No unscheduled deaths occurred in any study group. Severe clinical signs oftoxicity, decreased maternal body weights and body weight gains, a slight increase in postimplantation loss and a significantly decreased mean fetal weight were evident inthe50mg/kg/day dose group. A slight increase in fetuses with reduced ossification (associated with reduced fetal weights) and an increase in bent ribs (attributed to maternal toxicity in this group) were observed at the 50 mg/kg/day dose level. Scattered, infrequent clinical findings and a slightly reduced body weight gain over the treatment period (gestation days 6-16) occurred at the 25 mg/kg/day dose level.The 5 mg/kg/day group was comparable to the vehicle control group in all parametersmeasured. The infrequent occurrence and nature of the malformations were not indicative of a teratogenic response in any dose group.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Rodwell study is a reliable study with a klimosch score of 1.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Two studies, one in rats (Rodwell 1986) and one in mice (Yasuda 1980), are available for the evaluation of the effects of DPG on the development.

Potential maternal, embryotoxic and teratogenic effects of 1,3-diphenylguanidine were evaluated in rats (Rodwell, 1986). DPG was administered orally by gavage to three groups of 25 bred CD female rats as a single daily dose of 0, 5, 25 and 50 mg/kg/day from days 6 through 15 of gestation. Throughout gestation, all females were observed twice daily for toxicity and body weights were recorded at appropriate intervals. On day 20 of gestation, all surviving females were sacrificed for Cesarean section; foetuses were weighed, sexed and examined for external, skeletal and soft tissue anomalies and developmental variations.

No unscheduled deaths occurred in any study group. Severe clinical signs of toxicity, decreased maternal body weights and body weight gains, a slight increase in post-implantation loss and a significantly decreased mean foetal weight were evident in the 50 mg/kg/day dose group. A slight increase in foetuses with reduced ossification (associated with reduced foetal weights) and an increase in bent ribs (attributed to maternal toxicity in this group) were observed at the 50 mg/kg/day dose level. Scattered, infrequent clinical findings and a slightly reduced body weight gain over the treatment period (gestation days 6-16) occurred at the 25 mg/kg/day dose level. The 5 mg/kg/day group was comparable to the vehicle control group in all parameters measured. The infrequent occurrence and nature of the malformations were not indicative of a teratogenic response in any dose group.

In conclusion, DPG induced severe maternal toxicity at a dose level of 50 mg/kg/day. Fetotoxicity was also expressed at this dose level by a significantly reduced mean foetal body weight and by an increase in foetal variations.A dose level of 25 mg/kg/day was considered a marginal NOAEL for foetotoxic effect.

 

Groups of 20 pregnant mice of the ICR-JCL strain were given DPG orally in doses of 0.25, 1.0, 4.0, or 10.0 mg/kg of body weight/day throughout pregnancy (Yasuda et al., 1980). Control mice were fed the vehicle atone. On day 18 of pregnancy, all mice were killed and the foetuses were examined.

Disturbances in implantation were seen in the mothers treated with 10 mg/kg/day (the highest dose) of DPG. Retarded ossification of the talus was seen in the foetuses of mothers treated with 4.0 mg/kg/day, but there was no dose-response relationship to this finding. Although malformations such as open eyelids or polydactyly were seen sporadically, these were categorised as spontaneous anomalies. Thus, DPG seems to have no detrimental effects on the development of mouse foetuses in doses of 4 mg/kg or less. The NOAEL for teratogenicity was higher than 10 mg/kg/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Rodwell study is chosen as key study because it is the most recent and the most reliable study with a klimisch score of 1.

Justification for classification or non-classification

Mandatory classification

- Regulation (EC) No 1272/2008 Annex VI Table 3.1 : Repro. 2, H361f (Suspected of damaging fertility)

   

This classification was adopted by the Commission Working Group on the Classification and Labelling of Dangerous Substances in July 1997 (ECBI/32/97) based on sperm and fertility effects reported by Bempong.

Bempong analysed the effects of 1,3-diphenylguanidine (purity not reported, probably of low purity according to correspondence with the author) on seminal cytology, testicular development and fertility (Bempong, 1983a and 1983b; Bempong & Hall, 1983; reliability 3). Dose-levels of 4 and 8 mg/kg bw/day 1,3-diphenylguanidine, administered by the oral route up to 15 weeks, induced a time- and dose-dependent increase in the frequency of sperm abnormalities in both mice and hamsters from week 4, a significant decrease in sperm count and testes weight from week 5, and irregularly shaped seminiferous tubules in mice. The fertility index and the number of implants per pregnant female mice were decreased in a dose-dependent fashion, but the effect did not seem to be time-dependent. The frequency of early or late dead foetuses per litter was significantly increased at the 5th and 7th week of dosing at the high dose levels.

 

Taken into account the uncertainties related to the study protocol (lack of details on compound purity, mode of administration, number of treated animals/dose, food and water consumption, clinical signs and body weight, poor statistical evaluation and no GLP) and the conflicting results with the other available information, these studies were considered as not reliable (reliability 3) by the OECD experts (SIAM 14, March 2002) and not taken into account to evaluate the reprotoxicity of 1,3-diphenylguanidine. The difference in these results with more recent studies may be at least explainable by contamination of the 1,3-diphenylguanidine employed in the studies carried out by Bempong.

 

Self-classification

 

Taken into account the reliable studies, 1,3-Diphenylguanidine, with a purity representative of the industrial product, did not affect the fertility of male mice (Van Marwijk and Koëter, 1989;Koëter et al., 1992) and male and female rats (Davies, 2010) when administered by gavage up to the maximal tested dose level of 16 and 25 mg/kg/d, respectively. In addition to the results of the feeding sub-chronic studies on the rat and mouse (Irwin, 1995; Murata et al., 2000), special studies for recognising reproductive toxic effects were also performed (Irwin, 1995). Comparisons of the parameter changes with the results of tests with feed withdrawal infer that the effects observed in the 1,3-diphenylguanidine-treated animals in high concentration groups are a result of the poor general state of health (malnutrition, exhaustion) of the animals and not a direct toxic effect on the reproductive organs. Very conservative NOAELs, based on the effects on the reproductive organs, secondary to malnutrition and exhaustion, can be established at 32 mg/kg bw/d for rats and from 16 to 114 mg/kg bw/d for mice. Moreover no adverse effect on reproductive organs or fertility were observed in the recent screening study (Davies 2010) at the highest dose of 25 mg/kg/day.

 

Therefore, no classification for reproductive toxicity is warranted according to the criteria of Regulation (EC) No 1272/2008 and Directive 67/548/EEC.