Disodium phosphonate

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:

03/2015 to 12/2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to OECD guideline under GLP conditions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Disodium phosphonate

EC Number:

237-249-1

EC Name:

Disodium phosphonate

Cas Number:

13708-85-5

Molecular formula:

Na2HPO3

IUPAC Name:

disodium phosphonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Bruggolen® H10
- Substance type: inorganic
- Physical state: solid
- Lot/batch No.: 14060611
- Expiration date of the lot/batch: 29.02.2016
- Storage condition of test material: ambient conditions

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Lecco Italy
- Age at study initiation: 7-9 wks
- Weight at study initiation: (P) Males: 200-225 g; Females: 175-200 g
- Housing: polysulphone solid bottomed cages
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via
G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) was offered ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 2 °C
- Humidity (%): 55 +/- 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until positive identification occurs or 14 days have elapsed
- Proof of pregnancy: [vaginal plug] referred to as [day 0] of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulations of the test item were prepared as solutions in water. Concentration
was assessed for all levels by taking two analytical aliquots (approximately
1 mL). Each analytical aliquot was analysed separately. Concentration was
evaluated as the mean of the two determinations.

Duration of treatment / exposure:

Males:
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks
prior to pairing and thereafter through the day before necropsy.

Females:
Animals will be dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks
prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice.

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100 mg/kg bw
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
300 mg/kg bw
Basis:
nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

no

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before treatment and once daily during treatment

BODY WEIGHT: Yes
- Time schedule for examinations:
Males were weighed weekly from allocation to termination. Females were
weighed weekly from allocation to positive identification of mating and on
Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4
post partum.

Litter observations:

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 abormalities

GROSS EXAMINATION OF DEAD PUPS:
yes

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
The clinical history of the males and females of the parental generation was
studied and a detailed post mortem examination was conducted (including
examination of the external surface and orifices).


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively:

Adrenal glands
Brain (cerebrum, cerebellum, medulla/pons)
Clitoris
Epididymides
Kidneys
Liver
Ovaries with oviduct
Penis with preputial glands
Pituitary
Prostate gland
Spleen
Seminal vesicles with coagulating glands
Testes
Thymus (where present)
Thyroid
Uterus with cervix
Vagina

Postmortem examinations (offspring):

As soon as possible after parturition was considered complete (Day 0 or 1
post partum), all pups (live and dead) were counted, sexed and live pups were
identified. Live pups were individually weighed on Days 1 and 4 post partum.
Pups dying during the lactation period were weighed before the despatch to
necropsy. Observation was performed once daily for all litters.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables
the significance of the differences amongst group means was assessed by
Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the
other parameters. Intergroup differences between the control and treated
groups were assessed by the non-parametric version of the Williams test. The
criterion for statistical significance was p<0.05.

Reproductive indices:

Males: Copulation index and Fertility index
Females: Copulatory index and Fertility index, Pre-birth loss, Pup loss at birth, cumulative pup loss, pre-implantation loss
Males and females: Pre coital interval

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female dosed at 1000 mg/kg/day was humanely killed on Day 24 of
the gestation period for a prolapse of the uterus. Macroscopically and
microscopically, no lesions were noted.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

1 Mortality and fate of females:
One high dose female (no. X0170069) was humanely killed on Day 24 of
gestation period for prolapse of the uterus. No clinical signs were observed
in this animal during the study. At post mortem examination, unilateral
implantation was observed. Macroscopically and microscopically, no lesions
were noted.
A total of 10 females were proved to be not pregnant at necropsy: 2 females
in the control group, 2 in the low dose group, 3 in the mid-dose group (1
of these, No. X0170059, did not mate) and 3 in the high dose group. One
female from the low dose group (no. X0170023), one from the mid-dose
group (no. X0170041) and one from the high dose group (no. X0170069)
showed unilateral implantation, while mating was not detected in 1 female
(no. X0170003) of the control group.
The number of females with live pups on Day 4 post partum was: 8 in the
control group, 7 in each of the low and mid-dose groups, 6 in the high dose
group. Total litter loss was observed in a single female (no. X0170023) from
the low dose group.


2 Clinical signs:
No signs of toxicological relevance were observed during the study.


3 Body weight and body weight gain:
No significant changes in body weight were observed in the treated animals
when compared to controls.

4 Food consumption:
Food consumption was unaffected by treatment in both sexes during the
study.

5 Oestrus cycle, reproductive parameters, pairing combination
and mating performance:
No treatment-related anomalies were noted in the oestrus cycle of the treated
females when compared to controls. The mean pre-coital interval and the
number of copulation plugs were similar between control and treated groups.

All females mated with the exception of female no. X0170059 of the middose
group. However, 2 females in the control group (nos. X0170005 and
X0170017), 2 in the low dose group (nos. X0170025 and X0170039), 3 in
the mid-dose group (nos. X0170047, X0170057 and X0170059, one of which
did not mate) and 3 in the high dose group (nos. X0170061, X0170075 and
X0170079) were found not pregnant.
The copulatory index was 100% for both sexes for Groups 1, 2 and 4 and
90% for Group 3. The fertility indices were 80% in Groups 1 and 2, 70% in
Groups 3 and 4.


6 Implantation, pre-birth loss data and gestation length of
females:
Gestation periods were similar in treated groups and controls. All dams gave
birth between Days 22 and 23 post coitum (with the exception of animal
no. X0170049 who gave birth on Day 21). Corpora lutea, implantations and
pre-implantation loss, total litter size and pre-birth loss (percentage) did not
show dose-related or treatment-related differences.

7 Litter data at birth, on Day 1 and on Day 4 post partum
of females and sex ratio of pups:
No significant differences in total litter size, live litter size, mean pup loss,
sex ratio and mean pup weights were observed among the surviving treated
dams and the controls.

8 Clinical signs of pups:
No relevant clinical signs, that could be related to treatment, were reported
at the examination of pups during the lactation period.

9 Necropsy findings in decedent pups and in pups sacrificed
on Day 4 post partum:
No relevant differences between control and treated groups were noted at
necropsy in the decedent pups. No significant abnormalities were recorded in
the pups sacrificed on Day 4 post partum or in the pup humanely killed on
Day 1 post partum (animal no. X0170049, pup no. 17) due to the absence of
skin in the neck and ventral region.

10 Terminal body weight and organ weights:
Terminal body weight was unaffected by treatment in both sexes.
No changes of toxicological significance were observed in the weight of the
organs. The statistically significant increase of the relative weight of kidneys
observed in the high dose females was not considered to be of toxicological
relevance, since it was of low magnitude and not supported by histopatological
examination.

11 Macroscopic observations:
Detailed macroscopic observations have been reported for male and female
animals from all groups.
No relevant changes were noted in treated animals. The sporadic changes
observed in few treated animals could be considered incidental.

12 Microscopic observations:
Histopathological evaluation was performed on selected tissues/organs in
control and high dose groups, as well as on all abnormalities detected during
post mortem observation.
No treatment-related changes were noted.
A limited number of lesions, reported in control and treated animals, were
considered to be an expression of spontaneous and/or incidental pathology,
seen in this species and age of untreated animals.

Spermatogenic cycle:
Seminiferous tubules were evaluated with respect to their stage in the
spermatogenic cycle and to the integrity of the various cell types within
the different stages; regular layering in the germinal epithelium was noted.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

food efficiency

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

histopathology: neoplastic

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

No relevant differences between control and treated groups were noted at
necropsy in the decedent pups. No significant abnormalities were recorded in
the pups sacrificed on Day 4 post partum or in the pup humanely killed on
Day 1 post partum (animal no. X0170049, pup no. 17) due to the absence of
skin in the neck and ventral region.

Body weight and weight changes:

not examined

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:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Clinical signs of pups:
No relevant clinical signs, that could be related to treatment, were reported
at the examination of pups during the lactation period.

Necropsy findings in decedent pups and in pups sacrificed
on Day 4 post partum:
No relevant differences between control and treated groups were noted at
necropsy in the decedent pups. No significant abnormalities were recorded in
the pups sacrificed on Day 4 post partum or in the pup humanely killed on
Day 1 post partum (animal no. X0170049, pup no. 17) due to the absence of
skin in the neck and ventral region.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Executive summary:

The effects of the test item, Bruggolen® H10, on fertility, pregnancy and early lactation of the offspring were investigated when administered orally to male and female Wistar Hannover rats. Groups of 10 males and 10 females received the test item by gavage at dosages of 100, 300 and 1000 mg/kg/day. One female dosed at 1000 mg/kg/day was humanely killed on Day 24 of the gestation period for a prolapse of the uterus. Macroscopically and microscopically, no lesions were noted. No treatment-related effects were detected in males and in the remaining females from the high dose and the other treated groups during the in vivo phase. No treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls. Copulatory and fertility indices did not show any treatment-related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio did not show any changes of toxicological relevance. Necropsy findings in pups did not reveal any treatment-related effect. No relevant changes were detected at post mortem examination in treated animals, when compared with controls. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages and a regular layering in the germinal epithelium was described. In conclusion, no treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (100, 300 and 1000 mg/kg/day). No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated. On the basis of the results obtained in this study, the NOEL (No Observed Effect Level) for general toxicity and for fertility and reproduction parameters was considered to be 1000 mg/kg/day for males and females.

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