Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2015 - To be completed
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP- and OECD-test guideline compliant study.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-chloro-N,N-diethyl-1,1-diphenyl-1-(phenylmethyl)phosphoramine
EC Number:
411-370-1
EC Name:
1-chloro-N,N-diethyl-1,1-diphenyl-1-(phenylmethyl)phosphoramine
Cas Number:
82857-68-9
Molecular formula:
C23 H27 Cl N P
IUPAC Name:
benzyl(diethylamino)diphenylphosphanium chloride
Test material form:
other: White powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Males: 10 weeks; Females: 9 weeks
- Weight at study initiation: Males: approx. 400 g; Females: approx. 220 g
- Fasting period before study: No
- Housing: individually (except during mating and lactation) in cages (TEcniplast 2154: 940 cm², polycarbonate with stainless steel lid) containing autoclaved sawdust
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 8 to 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 08 September 2015 To: 15 November 2015

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Drinking water tretead by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: test item weighed and mixed with required amount of vehicle in accordance with internal procedures

VEHICLE
- Concentration in vehicle: 0, 2.5, 5, 8 or 10 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Details on mating procedure:
Females were paired with males at the same dose-level. One female was placed with one male, in the latter's cage, overnight.
Mating was confirmed in the morning by checking for the presence of a vaginal plug or for sperm in a vaginal lavage.
The day of confirmed mating was designated Day 0 p.c.
Each female was placed with the same male until mating occurs or 7 days have elapsed. Any pair with no evidence of mating after 7 days was separated and the female was placed for a further 7 days with a different male (at the same dose-level) that had already mated.
The pre-coital time was calculated for each female.
Any female with no evidence of mating after 7 days with the second male would have been sacrificed 24 to 26 days after the end of the mating period (unless delivery occurs).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analytical technique: High Performance Liquid Chromatography with UV detection (HPLC/UV).
- Principle and validation of the method: Analytical method was validated at CiToxLAB France (CiToxLAB France/Study No. 42844 VAA) before dose formulation analysis. Checked parameters, acceptance criteria and obtained results are described in the validation report.
- Determination of test item concentrations in dose formulations: Once in Weeks 1, 3/4 and 5/6 (three occasions). Samples of control and test item dose formulations were analyzed using the validated method. Acceptance criterion: Measured concentration = nominal concentration ± 10%.
Duration of treatment / exposure:
The dose formulations will be administered daily according to the following schedule:

Males:
- 2 weeks before mating,
- during the mating period (up to 2 weeks),
- until sacrifice (at least 4 weeks in total),

Females:
- at least 2 weeks before mating,
- during the mating period (up to 2 weeks),
- during gestation,
- during lactation until Day 4 p.p. (post partum) inclusive,
- until sacrifice for females with no evidence of mating or no delivery.

Day 1 corresponds to the first day of the treatment period.
Frequency of treatment:
See above
Details on study schedule:
See above
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 25, 50 and 100 mg/kg bw/day
Basis:
nominal in water
From Study Day 1 to 18
Remarks:
Doses / Concentrations:
0, 25, 50 and 80 mg/kg bw/day
Basis:
nominal in water
From Study Day 19 on (due to mortality in high-dose group)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection:

The initial dose-levels were based on the results of the following previous studies:

a) 4-week toxicity study (1991): GM 102 E was administered daily by oral gavage to Sprague Dawley rats (5 rats/sex/group) at dose-levels of 6.25, 12.5, 25 and 50 mg/kg/day. The formulations were administered as solutions in water (10 mL/kg/day). Ptyalism (hypersalivation) was observed immediately after dosing. From study Week 3 onwards, there were no noteworthy findings. Under the condition of this study the No Observed Effect Level (NOEL) was considered to be 50 mg/kg/day.

b) Acute toxicity studies (1983 and 1994): two studies were conducted by the oral route (gavage) in two different strains of rats. The LD50 values were the following:
- 160 mg/kg, 95% confidence interval: [123-208], Wistar rats (1983),
- 123 mg/kg, 95% confidence interval: [100-150], Sprague-Dawley rats (1994).

The high dose-level was set at 100 mg/kg/day. The low and intermediate dose-levels were selected using a ratio representing a 2-fold interval (i.e. 25 and 50 mg/kg/day). Due to elevated mortality in the high-dose group, the dose of 100 mg/kg/day was reduced to 80 mg/kg/day from Study Day 19 on.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
- Morbidity and mortality:
Each animal was checked for mortality and morbidity at least once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays. Any animal showing signs of poor clinical condition, especially if death appeared imminent, was humanely sacrificed. A macroscopic post-mortem examination was performed on any animals found dead or prematurely sacrificed.

- Clinical signs:
From arrival, each animal was observed at least once a day as part of routine examinations. From the start of the treatment period, each animal was observed at least once a day, at approximately the same time of day, for the recording of clinical signs.

- Body weight:
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), once a week until mated (or until sacrifice for females with no evidence of mating), then on Days 0, 7, 14 and 20 p.c. (post-coitum) and on Days 1 and 5 p.p.

- Food consumption:
The quantity of food consumed by each male was measured once a week, over 7-day periods, from the first day of treatment until the start of the mating period. The quantity of food consumed by each female was measured once a week, over 7-day periods, from the first day of treatment until the start of the mating period, then during gestation over 7 day periods (Days 0-7, 8-14 and 15-20 p.c.), and during lactation (Days 1-5 p.p.). During the mating period, food consumption was not measured for males or females. Food intake per animal and per day was calculated by noting the difference between the food given and that remaining in the food-hopper at the next distribution.
Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period until the females were mated.
Litter observations:
Each live pup was individually identified on Day 1 post-partum (morning of complete parturition), by subcutaneous injection of indian ink.

- Litter size:
The total litter size and the sex of each pup was recorded as soon as possible after birth. Any gross external malformations in pups was noted. Litters were observed daily to note the number of live, dead and cannibalized pups.

- Clinical signs:
The pups were observed daily for clinical signs, abnormal behavior and external abnormalities.

- Body weight:
The body weight of each pup was recorded on Days 1 and 5 p.p.
Postmortem examinations (parental animals):
On completion of the treatment period, all surviving F0 animals were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination:
- Males: after the end of the mating period (at least 4 weeks of treatment in total),
- Females: on Day 5 p.p.,
- Females which did not deliver: on Day 24-26 p.c. (after a body weight recording to check for a possible un-noticed delivery; sacrifice by inhalation of carbon dioxide gas followed by cervical dislocation was used if gestation was suspected),
- Females with no evidence of mating: 24-26 days after the end of the mating period if no delivery occurred (sacrifice by inhalation of carbon dioxide gas followed by cervical dislocation was used if gestation was suspected),
- Females with total litter loss: as appropriate.

--> Animals prematurely sacrificed or found dead
- F0 Males:
Prematurely sacrificed males were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. A complete macroscopic post mortem examination was performed on any males found dead or prematurely sacrificed.

- F0 Females:
During the pre-mating period: Prematurely sacrificed females were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. A complete macroscopic post mortem examination was performed on any females found dead or prematurely sacrificed.

During the mating and gestation periods: During the mating period, prematurely sacrificed females were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination, except if gestation was suspected (see below). During the gestation period, prematurely sacrificed females were euthanized by inhalation of carbon dioxide gas followed by cervical dislocation. A complete macroscopic post mortem examination was performed on any females found dead or prematurely sacrificed.
The pregnancy status was determined and, if appropriate and possible, the numbers of corpora lutea and implantation sites was recorded and classified as live or dead concepti, early or late resorptions or scars. For apparently non-pregnant (mated or apparently unmated) females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964), except between Days 0 and 5 p.c. inclusive.

During the lactation period: Prematurely sacrificed females were deeply anesthetized by intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination. A complete macroscopic post mortem examination was performed on any females found dead or prematurely sacrificed. In addition, the number of corpora lutea and implantation sites was recorded.

The body weight of each F0 animal sacrificed as scheduled (after the end of the mating period for males or on Day 5 p.p. for females) was recorded before sacrifice. The organs selected for microscopic examination were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all F0 animals, including any that died during the study or were sacrificed prematurely. Particular attention was accorded to the reproductive organs. The number of corpora lutea and implantation sites was recorded for females sacrificed as scheduled on Day 5 p.p. and, if possible, for any females sacrificed 24 to 26 days after the end of the mating period with no evidence of mating and for any females sacrificed on Day 24-26 p.c. due to no delivery. For apparently non-pregnant females the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique.

The following tissues were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in Modified Davidson's Fixative): Macroscopic lesions, Epididymides, Mammary gland area, Ovaries (with oviducts), Prostate, Seminal vesicles (with coagulating glands), Testes, Uterus (horns and cervix), Vagina. All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately 4 microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS).

Microscopic examination was performed on the reproductive organs from control, mid- and high-dose males and females at terminal sacrifice and from premature decedents, and on all macroscopic observations in all groups. Particular attention was accorded to stages of spermatogenesis in male gonads and histopathology of interstitial testicular cell structure.



Postmortem examinations (offspring):
Pups were sacrificed by intraperitoneal injection of sodium pentobarbital:
- Pups whose mother dies: as soon as possible,
- Surviving pups: on Day 5 p.p.

Moribund pups and pups prematurely sacrificed because their dam died were sacrificed by intraperitoneal injection of sodium pentobarbital. A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all found dead and prematurely sacrificed pups. Particular attention was accorded to the reproductive organs and to whether the pup had fed (e.g. presence of milk in the stomach). No tissues were preserved.

A macroscopic post-mortem examination of the principal thoracic and abdominal organs was performed on all pups sacrificed on Day 5 p.p. No tissues were preserved.






Statistics:
- Body weight, food consumption and reproductive data:
Data were compared by one-way analysis of variances and the Dunnett test (where mean values are considered as normally distributed, and variances are considered as homogenous) or by Fisher’s exact probability test (proportions).

- Organ weights:
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
Reproductive indices:
- Pre-implantation loss: (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea x 100
- Post-implantation loss (calculated manually): (Number of implantation sites - Number of live pups) / Number of implantation sites x 100 - Mating index: Number of mated animals / Number of paired animals x 100
- Fertility index: Number of pregnant female partners / Number of mated pairs x 100
- Gestation index: Number of females with live born pups / Number of pregnant females x 100
Offspring viability indices:
- Live birth index: Number of live born pups / Number of delivered pups x 100
- Viability index on Day 4 p.p.: Number of surviving pups on Day 4 p.p. / Number of live born pups x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Mortality in high-dose group. Severe clinical signs in mid- and high-dose groups.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Lower mating and fertility indexes in high-dose group.

Details on results (P0)

- Mortality:
There were no unscheduled deaths in the control, 25 or 50 mg/kg/day groups. Test item treatment related deaths were recorded in 2/10 males and 8/10 females from the 100/80 mg/kg/day group. Before sacrifice for ethical reason or in the found dead rats, piloerection, round back, emaciated appearance, hypoactivity, loud breathing, abdominal breathing, dyspnea, ptyalism, half closed eyes and/or chromorhinorrhea were observed in most animals. Changes were observed in the lungs, spleen, stomach, ileum, cecum and/or vagina and these findings were considered to be agonal and/or to have contributed to the death or the moribund status of these animals, but the cause of death could not be ascertained.

- Clinical signs:
In the 100/80 mg/kg/day group, piloerection and round back were considered to be test-item treatment related and adverse. In the 50 mg/kg/day, round back was considered to be test-item treatment related and adverse. At 25 mg/kg/day, there were no adverse findings. Ptyalism was considered to be related to the treatment with the test item but of minor toxicological significance.

- Body weight and mean body weight change:
When compared with controls, there were no adverse effects on mean body weight or mean body weight change, both in males and females.

- Food consumption:
When compared with controls, there were no adverse effects on mean food consumption.

- Mating and fertility data:
There were no effects on the mean number of days taken to mate (i.e. pre-coital time). When compared with control, there were lower mating and fertility indexes at 100/80 mg/kg/day. At these dose-levels, mating and/or fertility indexes were below the lower limit of the Historical Control Data. Therefore these findings were considered to be test-item treatment related and toxicologically significant. There were no adverse effects at 25 and 50 mg/kg/day.

- Gestation and Delivery data:
When compared with controls, there were no effects on mean duration of gestation and no adverse effects at 25 mg/kg/day on gestation and delivery data. At 100/80 mg/kg/day, the severe mortality and the limited number of dams makes irrelevant any comparison with the control group and Historical Control Data. At 50 mg/kg/day and when compared with controls, there was a low mean number of pups delivered at birth (10.9 vs. 14.0, p<0.05). At this dose-level the mean number of pups delivered was also below the lower limit of the Historical Control Data. This finding was considered to be related to the test-item treatment and adverse.

- Pathology:
There were no organ weights changes related to the test item. There were no macroscopic or microscopic changes noted in the testes, epididymides and ovaries in animals treated at 100/80 mg/kg/day or at 50 mg/kg/day.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
50 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality
Dose descriptor:
NOAEL
Remarks:
Reproductive performance (mating, fertility and delivery data)
Effect level:
25 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: lower number of pups delivered

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Marked increase in pups cold to the touch and with a few milk in the stomach as well as in the incidence of litters affected in high-dose group.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Marked increase in the number of found dead, moribund and cannibalized pups as well as in the incidence of litters affected in high-dose group.
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

- Pups mortality:
At 100/80 mg/kg/day and when compared with controls there was a marked increased in the number of found dead, moribund and cannibalized pups as well as in the incidence of litters affected (5/6 vs. 3/10). These findings were considered to be test item treatment related and adverse.

- Pups clinical signs and external abnormalities:
At 100/80 mg/kg/day and when compared with controls there was a marked increase in pups cold to the touch and with a few milk in the stomach as well as in the incidence of litters affected (3/6 vs. 2/10). These findings were considered to be the consequence of abnormal maternal behavior, to be test item treatment related and adverse. There were no external abnormalities.

- Pup viability:
There were no effects on the live birth, viability and lactation indexes up to 50 mg/kg/day.

- Pup body weight:
There were no adverse effect on mean pup body weight and mean pup body weight change.

- Pup sex ratio:
There were no effects on the mean percentage of male fetuses (sex-ratio).

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
Toxic effects on progeny
Generation:
F1
Effect level:
50 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: low pup viability

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

- Test item concentrations:

Dose formulations analyzed in weeks 1, 3 and 6 remained within an acceptable range of -6.1% to +5.3% when compared to the nominal values ( ± 10% of the nominal concentrations).

On weeks 1 and 3, no test item was observed in the control dose formulation. On week 6, a peak was observed at the test item retention time. However, it was below the limit of quantification (< 0.0001 mg/mL) and was considered as negligible.

Applicant's summary and conclusion

Conclusions:
- The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 50 mg/kg/day (based on mortality and clinical recorded for both sexes at 100/80 mg/kg/day),
- The NOAEL for reproductive performance (mating, fertility and delivery data) was considered to be 25 mg/kg/day (based on the lower number of pups delivered from 50 mg/kg/day),
- The NOAEL for toxic effects on progeny was considered to be 50 mg/kg/day (on the low pup viability at 100/80 mg/kg/day).
Executive summary:

GM 102 E (batch No. 180020) was administered daily by oral gavage to male and female Sprague‑Dawley rats, for 2 weeks before mating, during mating, and until sacrifice (for males) or throughout gestation and until day 4post-partum(for females), at dose-levels of 25, 50 or 100/80 mg/kg/day.

Animals were checked daily for clinical signs and mortality. Body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5p.p.. The total litter sizes and numbers of pups of each sex were recorded after birth. The pups were observed daily for clinical signs of toxicity and pup body weights were recorded on days 1 and 5p.p.. The males were sacrificed after completion of the mating period. Dams were sacrificed on day 5 p.p.. Body weights and selected organs weights were recorded and a complete macroscopicpost-mortemexamination performed, with particular attention paid to the reproductive organs. Microscopic examination was performed on the reproductive organs from control, mid- and high-dose males and females at terminal sacrifice and from premature decedents, and on all macroscopic observations in all groups. Pups, including those found dead before study termination, were also submitted for a macroscopic post-mortem examination.

In the parental generation, there were no unscheduled deaths in the control, 25 or 50 mg/kg/day groups. Test item treatment related deaths were recorded in 2/10 males and 8/10 females from the 100/80 mg/kg/day group. Before sacrifice for ethical reason or in the found dead rats, piloerection, round back, emaciated appearance, hypoactivity, loud breathing, abdominal breathing, dyspnea, ptyalism, half closed eyes and/or chromorhinorrhea were observed in most animals. Changes were observed in the lungs, spleen, stomach, ileum, cecum and/or vagina and these findings were considered to be agonal and/or to have contributed to the death or the moribund status of these animals, but the cause of death could not be ascertained. In the 100/80 mg/kg/day group, piloerection and round back were considered to be test-item treatment related and adverse. In the 50 mg/kg/day, round back was considered to be test-item treatment related and adverse. At 25 mg/kg/day, there were no adverse findings. Ptyalism was considered to be related to the treatment with the test item but of minor toxicological significance.

There were no effects on the mean number of days taken to mate (i.e.pre-coital time). When compared with control, there were lower mating and fertility indexes at 100/80 mg/kg/day. At these dose-levels, mating and/or fertility indexes were below the lower limit of the Historical Control Data. Therefore these findings were considered to be test-item treatment related and toxicologically significant. There were no adverse effects at 25 and 50 mg/kg/day. When compared with controls, there were no effects on mean duration of gestation and no adverse effects at 25 mg/kg/day on gestation and delivery data. At 100/80 mg/kg/day, the severe mortality and the limited number of dams makes hazardous any comparison with the control group and Historical Control Data. At 50 mg/kg/day and when compared with controls, there was a low mean number of pups delivered at birth (10.9 vs. 14.0, p<0.05). At this dose-level the mean number of pups delivered was also below the lower limit of the Historical Control Data. This finding was considered to be related to the test-item treatment and adverse.

In the offspring, at 100/80 mg/kg/day and when compared with controls there was a marked increased in the number of found dead, moribund and cannibalized pups as well as in the incidence of litters affected (5/6 vs. 3/10). These findings were considered to be test item treatment related and adverse. At 100/80 mg/kg/day and when compared with controls there was a marked increase in pups cold to the touch and with a few milk in the stomach as well as in the incidence of litters affected (3/6 vs. 2/10). These findings were considered to be the consequence of abnormal maternal behavior, to be test item treatment related and adverse. There were no external abnormalities.

Based on the experimental conditions of this study:

- The No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 50 mg/kg/day (based on mortality and clinical recorded for both sexes at 100/80 mg/kg/day),

- The NOAEL for reproductive performance (mating, fertility and delivery data) was considered to be 25 mg/kg/day (based on the lower number of pups delivered from 50 mg/kg/day),

- The NOAEL for toxic effects on progeny was considered to be 50 mg/kg/day (on the low pup viability at 100/80 mg/kg/day).