N-(2-nitrophenyl)phosphoric triamide

Toxicity to reproduction

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:

2012-07-30 - 2013-02-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The GLP study was conducted according to an internationally accepted guideline. All study parameters are based on the specific guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

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

Test material information

Constituent 1

Reference substance name:

-

EC Number:

477-690-9

EC Name:

-

Cas Number:

874819-71-3

Molecular formula:

Hill formula: C6H9N4O3P CAS formula: C6H9N4O3P

IUPAC Name:

N-(diaminophosphoryl)-2-nitroaniline

Test material form:

solid: crystalline

Details on test material:

Designation: N-(2-nitrophenyl)phosphoric triamide
CAS no.: 874819-71-3
EC no.: 477-690-9
Batch no.: P101/04/S-11
Characteristics: Yellow powder.
Content: 98.6%

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species / Strain / Stock: Rat / CD / Crl:CD(SD)
Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Age of animals (at first dosing): Males: 55 days; Females: 55 days
Body weight (at first dosing): Males: 325.7 - 365.5 g; Females: 183.9 - 219.1 g
Adaptation period: 5 days

Housing
Except during the mating period, the males and females were kept singly in MAKROLON cages (type III plus) at a room
temperature of 22°C ± 3°C (maximum range) and a relative humidity of 55% ± 15% (maximum range). Deviations from the maximum range caused for
example during cleaning procedures are dealt with in SOPs. The rooms were alternately lit (from 150 lux at 1.5 m room height) and darkened
for periods of 12 hours. Granulated textured wood (Granulat A2, Brandenburg, 49424 Goldenstedt- Arkeburg, Germany) was used as bedding material. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA1 is conducted at least once a year by LUFA-ITL.

Drinking water
Tap water was offered ad libitum.

Diet
Commercial ssniff R-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany, see Appendix 2 'Composition of the diet') served as food. The food was offered ad libitum. Food residue was removed and weighed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.8% aqueous hydroxypropylmethycellulose gel

Details on exposure:

Route of administration: Oral, via gavage.
Frequency of administration: Once daily.
Administration volume: 10 mL/kg b.w.
Duration of administration:
Males:
Once daily for 32 days (beginning 2 weeks
prior to mating lasting up to the day before
sacrifice until a minimum dosing period of 28
days was completed).
Females:
Once daily, beginning 2 weeks prior to mating
and continuing up to, and including, day 3
post-partum or the day before sacrifice.

Details on mating procedure:

Four (4) groups of sexually mature male and female rats were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period.
The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. If findings were negative, mating was repeated.
The day of conception (day 0 of gestation) was considered to be the day on which sperm was found. This procedure was repeated until at least 8 pregnant dams were available for each group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test item formulation analysis
For the analysis of the test item formulations, samples of approximately 10 mL
were taken at the following times and stored at -20°C or colder until analysis:
At study initiation: Analysis of concentration
Immediately after preparation of the solution as well as after 8 and 24 hours of storage of the test item preparations at room temperature.
(3 samples per dose level group)
Number of samples: 9

Analysis of homogeneity
At start of administration, during (middle) administration and before administration to the last animal of each dose level group.
(3 samples per dose level group)
Number of samples: 9

At termination of the administration period at a time point when the majority of dams were dosed:
Analysis of concentration
During the last administration of the test item to the group, always before administration to the last animal per dose level group.
(1 sample per dose level group)
Number of samples: 3
Sum of all samples: 21

The samples were labelled with the study number, species, type of sample, concentration, sampling time and date.

Duration of treatment / exposure:

Males:
Once daily for 32 days (beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed).

Females:
Once daily, beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Frequency of treatment:

once daily

Details on study schedule:

Duration of administration Males
Once daily for 32 days (beginning 2 weeks prior to mating lasting up to the day before sacrifice until a minimum dosing period of 28 days was completed).
Females
Once daily, beginning 2 weeks prior to mating and continuing up to, and including, day 3 post-partum or the day before sacrifice.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

45 mg/kg bw/day (actual dose received)

Remarks:

Doses / Concentrations:
0, 45, 135, 450 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Dose / conc.:

135 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

450 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels have been selected based on the results of a 14-day dose-range finding study:
In this study, animals were dosed with 100, 300 or 1000 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day.
None of the animals died prematurely.
Extremely yellow discoloured urine - caused by the inherent colour of the test item - and pilo-erection were noted starting at a dose level of 300 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day. In addition, some of the high dose rats showed pale faeces.
The body weight of the male rats treated orally with 300 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day was decreased by 10% on test day 15 and the body weight of the male and female rats treated orally with 1000 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day was reduced by up to 23 % for the males and by up to 11 % for the females on test days 8 and 15 compared to the control group. Body weight gain was reduced accordingly.
The relative food consumption of the male and female rats treated orally with 1000 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day was reduced by up to 15 % for the males and by up to 20 % for the females starting in test week 1.
At necropsy, yellow stomach content and extremely yellow discoloured urine were observed for all male and female animals treated with 1000 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day.

Examinations

Parental animals: Observations and examinations:

Clinical signs
Throughout the test period, each animal was observed individually for clinical signs at least once daily. The frequency was increased when signs of toxicitiy were observed.

Mortality
All animals were checked daily for viability early in the morning and again as late as possible in the afternoon.

Body weight
Males and females were weighed on the first day of dosing, weekly thereafter and at termination. During gestation, females were weighed on days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 or 1 post-partum) and day 4 postpartum. Body weights were recorded individually for each adult animal.

Food consumption
Food consumption of each animal was recorded weekly during the pre-mating period, daily during gestation and on day 4 post-partum.

Oestrous cyclicity (parental animals):

The duration of gestation was recorded and was calculated from day 0 of pregnancy.

Sperm parameters (parental animals):

Detailed histopathologic examination was performed on the ovaries, testes and epididymides (with special emphasis on the qualitative stages of spermatogenesis and histopathology of intestitial testicular structure) of the adult animals of the control and the high dose group following haematoxylin-eosin and PAS staining (testes and epididymides).

Litter observations:

As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
Live pups were counted and sexed within 24 hours of parturition (day 0 or 1 postpartum) and on day 4 post partum. Any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

The male animals were sacrificed after the end of the mating period on test day 33. Dams with offspring were sacrificed on day 4 post-partum or shortly thereafter.At the time of sacrifice, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded in the female adult animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI. The testes and epididymides of all male adult animals were weighed. The ovaries (2), testicles (2), epididymis (2), accessory sex organs (coagulating gland, preputial gland, prostate, seminal vesicle, uterus (incl. cervix and oviducts), vagina) and all organs showing macroscopic lesions of all adult animals were preserved. The testes and epididymides were preserved in Bouin's fixative. The remaining tissues were preserved in 7% buffered formalin.
Detailed histopathologic examination was performed on the ovaries, testes and epididymides (with special emphasis on the qualitative stages of spermatogenesis and histopathology of intestitial testicular structure) of the adult animals of the control and the high dose group following haematoxylin-eosin and PAS staining (testes and epididymides).

Postmortem examinations (offspring):

All pups sacrificed at day 4 post-partum and all prematurely deceased pups (pups found dead in the cage) were carefully examined externally for gross abnormalities.

Statistics:

The test item groups 2 to 4 were compared to the control group 1.
The following statistical methods were used:
For all numerical values homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p ≤ 0.01.
For the comparison of classification measurements the following statistical methods were employed (the limits of significance were p ≤ 0.05 and p ≤ 0.01):
­ FISHER's exact test, n < 100
or
­ chi2-test with Yates' correction for continuity, n ≥ 100
All data were evaluated statistically in this manner. In tables in which individual results differ significantly from those of the control group, these data are indicated.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations of the last decimal place of up to one may occur caused by rounding.

Reproductive indices:

The duration of gestation was recorded and calculated from day 0 of pregnancy. As soon as possible after delivery, each litter was examined to establish the
number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the
presence of gross abnormalities.
Live pups were counted and sexed within 24 hours of parturition (day 0 or 1 postpartum) and on day 4 post partum. Any abnormal behaviour of the offspring was recorded.
The following parameters were determined:
- Number of pregnant females
- Pre-coital time
- Gestation length
- Corpora lutea: number per dam
distribution in the uterine horns
absolute number per group
mean per group
- Implantation sites: number per dam
distribution in the uterine horns
absolute number per group
mean per group
Number of pups absolute: at birth (alive and dead), after 4 days of life
- Number of pups per dam (male/female/total): at birth, after 4 days of life
- Number of stillbirths: absolute, per dam
- Number of pups with malformations: absolute, per dam

Offspring viability indices:

Birth Index, Live Birth Index, Viability Index, Pre-implantation loss, Post-implantation loss

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

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):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Treatment with 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day led to an extreme yellow discoloured urine in all 10 high dosed males from test day 2 onwards.
The faeces of all males were of normal consistency throughout the experimental period.
Treatment with 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day led to an extreme yellow discoloured urine in all 10 high dosed females from test day 2 onwards.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

Details on results (P0)

Mortality:
None of the male or female animals died prematurely during the course of the study.

Clinical signs:
Treatment with the high dose of 450 mg N-(2- nitrophenyl)-phosphoric triamide/kg b.w./day led to an extreme yellow discoloured urine in all 10 high dosed males from test day 2 onwards. Treatment with 135 mg N-(2-nitrophenyl)- phosphoric triamide/kg b.w./day led to an extreme yellow discoloured urine in 4 of 8 pregnant females, noted to start on gestation day 19 in one female animal. From gestation day 21 onwards, all 4 dams were affected.
Treatment with 450 mg N-(2-nitrophenyl)- phosphoric triamide/kg b.w./day led to an extreme yellow discoloured urine in all 10 high dosed males from test day 2 onwards.

Body weight:
The body weight of the male animals treated with 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day was considerably below the body weight of the control group from approximately test day 8 onwards by up to 14%.
The body weight of the female animals treated with 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day was considerably below the body weight of the control group from gestation day 0 onwards by up to 20%.

Food consumption
Treatment with the high dose of 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day led to a slightly decreased food intake in gestation week 3 (gestation days 14 to 20) compared to the control group.

Reproduction data
Treatment with the high dose of 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day resulted in the following test item-related changes: a slightly reduced mean and total number of corpora lutea, implantation sites and, subsequently, a reduced mean number of pups at birth. An increased number of stillbirths resulted in a slightly increased postimplantation loss of 16% compared to the control group. Subsequently, a reduced mean
number of live born pups and a reduced live birth index of 91% (compared to 99% in the control group) were noted. The total number of live born pups was reduced accordingly.

Gross pathology
The macroscopic inspection of the male animals revealed a reduced size of the testes in 1 of 10 low dosed males, and in all 10 intermediate and high dosed male rats treated with either 45, 135 or 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day.
Treatment with either 45, 135 or 450 mg N-(2- nitrophenyl)phosphoric triamide/kg b.w./day led to dose-related decreased absolute epididymides
weights compared to the control group. In addition, decreased absolute testes weights were noted for the male rats of the intermediate and high dose group treated with 135 or 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day.

Histopathology
The microscopic changes observed in the testes of group 4 animals treated with 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./ day consisted of testicular atrophy with related tubular atrophy of the seminiferous tubules. Tubular damage was characterized by presence of interstitial oedema, loss of germ cell layers in the seminiferous tubules. The changes were characterized by moderate tubular atrophy with damage (degeneration/necrosis) of the germinal epithelium with sometimes 'Sertoli only' like appearance. There was loss of spermatogonia, spermatocytes, spermatids and spermatozoa reflecting damaged spermatogenesis/spermiogenesis. These test item-related testicular changes associated with the reduced size of
the testes as observed at macroscopy led to aspermia with only empty duct(s) or ducts containing cellular debris in the epididymides.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

reproductive performance

other: see 'Remark'

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

135 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: slightly reduced mean and total number of corpora lutea, implantation sites, reduced mean number of pups at birth, increased number of stillbirth at 450 mg/kg bw

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

135 mg/kg bw/day (actual dose received)

System:

male reproductive system

Organ:

testes

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

Mortality
A total of 55 pups of the high dose group were found dead or were cannibalised on lactation days 1 to 4 compared to 17 deceased or cannibalised pups in the control group. Subsequently, a severely reduced viability index of only 30% was calculated for the high dose group compared to a viability index of 88% in the control group (statistically significant at p ≤ 0.01).
The number of live male and female pups on lactation day 4 was accordingly severely reduced in the high dose group compared to the control (statistically significant at p ≤ 0.01 for males, females and male/female pups combined).

Body weight
Treatment of the parental animals with the high dose of 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day resulted in a reduced mean and total body weight of the male and female pups on lactation day 4.In addition, the total body weight of the pups of the high dose group was already
reduced on lactation day 1 compared to the control due to the low number of live born pups (male pups: by 34%, female pups: by 41% and male/female pups combined by 38% (statistically significant at p ≤ 0.01)).

Terminal external examinations
The external examinations of all prematurely deceased pups and of all pups at dissection on day 4 post-partum did not reveal any externally visible abnormalities, neither for the pups of the control group nor for any pup of the test item groups after treatment of the parental animals with 45, 135 or 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

135 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: severely increased number of deceased pubs, reduced mean and total body weight of the pubs at 450 mg/kg bw

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

450 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Changes in absolute epididymides and testes weights compared to the control at terminal sacrifice on test day 33 [%]
Organ	Group 2 45 mg/kg	Group 3 135 mg/kg	Group 4 450 mg/kg
	males	males	males
Epididymidis, left	- 14**	- 43**	- 46**
Epididymidis, right	- 11	- 42**	- 45**
Testis, left	none	- 53**	- 59**
Testis, right	none	- 52**	- 60**

Viability of F1pups during the first 4 lactation days
Parameter	Group 1 (Control)	Group 2 (45 mg/kg)	Group 3 (135 mg/kg)	Group (450 mg/kg)
Number of deceased pups during the first 4 lactation days	17 (4)^#	2	2	55
Viability index (%)	88.3 (97.0)^#	98.4**	98.5**	29.6**

Changes in mean and total body weights on lactation day 4 compared to the control [%]
Parameter	Group (450 mg/kg)
	Male pups	Female pups	Male and female pups combined
Mean body weight	- 26	- 23**	- 22
Total body weight	- 62	- 64**	- 61**

Applicant's summary and conclusion

Conclusions:

The following no-observed-adverse-effect levels were noted:

Effects on the parental animals

NOAEL (no-observed-adverse-effect level):

45 mg N-(2-nitrophenyl)phosphoric triamide/ kg b.w./day, p.o.

Effects on reproductive toxicity

NOAEL (no-observed-adverse-effect level):

135 mg N-(2-nitrophenyl)phosphoric triamide/ kg b.w./day, p.o.

Effects on the F1 pups

NOAEL (no-observed-adverse-effect level):

135 mg N-(2-nitrophenyl)phosphoric triamide/ kg b.w./day, p.o.

Executive summary:

The aim of the experiment was to obtain information on possible effects of the test item N-(2-nitrophenyl)phosphoric triamide on reproduction and/or development. The test item was administered orally to rats at dose levels of 45, 135 and 450 mg/kg b.w./day during the pre-mating, mating and post-mating periods to parental males as well as during the pre-mating, mating, gestation and lactation periods until day 3 post-partum to parental female animals.

Effects on the parental generation

None of the adult animals died prematurely. Starting at the low dose of 45 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day, marginal changes were already noted in form of decreased absolute epididymides weights. Further signs of systemic toxicity were noted from the intermediate dose of 135 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day onwards in form of an extreme yellow discoloured urine and decreased absolute testes weights. Treatment with the high dose of 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day caused additional changes in form of salivation, ataxia, pilo-erection and clonic-tonic convulsions, a reduced body weight and a decreased food intake. The macroscopic inspection at the end of the treatment period revealed changes in form of a reduced size of the testes in 1 of 10 low dosed males, and in all 10 intermediate and high dosed male rats. The histopathological examination of the parental animals of the high dose group revealed changes in the testes (e.g. testicular atrophy with related tubular atrophy of the seminiferous tubules, interstitial oedema, loss of germ cell layers in the seminiferous tubules, moderate tubular atrophy with damage (degeneration/necrosis) of the germinal epithelium, loss of spermatogonia, spermatocytes, spermatids and spermatozoa) and epididymides (aspermia with only empty duct(s) or ducts containing cellular debris).

Effects on the reproduction

No test item-related influence was noted on any of the reproduction parameters up to a dose level of 135 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day.

Treatment with the high dose of 450 mg N-(2-nitrophenyl)phosphoric triamide/kg b.w./day resulted in the following test item-related changes: a slightly reduced mean and total number of corpora lutea, implantation sites and, subsequently, a reduced mean number of pups at birth. An increased number of stillbirths resulted in a slightly increased post-implantation loss of 16% compared to the control group. Subsequently, a reduced mean number of live born pups) and a reduced live birth index of 91% (compared to 99% in the control group) were noted. The total number of live born pups was reduced accordingly.

Effects on the F1 pups

Treatment of the parental animals with the high dose of 450 mg N-(2-nitrophenyUphosphoric triamide/kg b.w./day resulted in a severely increased number of deceased pups during the first 4 lactation days and a reduced mean and total body weight of the male and female pups.