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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 January 2012 - 22 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Sodium Hypophosphite
- Physical state: white crystalline solid
- Lot/batch No.: 90113D
- Analytical purity: 99.5%
- Expiry date: 31 December 2012
- Storage conditions: at room temperature, in a well ventilated place, stored in a tightly closed container, the opening of the container being restricted to the minimum needs.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France.
- Age at study initiation: approximately 9-11 weeks old on the first day of treatment
- Mean body weight at the first day of treatment, i.e day 6 : 279 g (a range of 224 g to 350 g)
- Fasting period before study: no
- Housing: polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: a period of 4 or 5 days before the beginning of the treatment period.

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

IN-LIFE DATES: 12 February 2012 to 07 March 2012.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified water obtained by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose-levels were calculated in terms of sodium hypophosphite monohydrate (CAS 10039 56 2, MW 103.977 g/mol) which is the form supplied by the Sponsor.
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.
The test item dose-formulations were prepared on a weekly basis based on available stability data and were stored in brown glass bottles at room temperature "prior to use".

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-IC.
Before the start of treatment the suitability of the proposed preparation process was confirmed by analysis of the stability.
In the stability study, a range of dose-formulations were prepared at levels which cover the lowest and highest concentrations proposed for use in the study, and then stored at room temperature with no specific protection against light or humidity.
Results indicated satisfactory stability of the dose-formulations after 9 days storage at room temperature.
Details on mating procedure:
- Impregnation procedure: purchased time pregnant
- Proof of pregnancy: Detection of vaginal plug
Duration of treatment / exposure:
day 6 to day 20 post-coitum
Frequency of treatment:
Daily
Duration of test:
21 days
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
24 mated females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of a preliminary 2 week toxicity study in the rat and on the basis of a combined repeated dose study with the reproduction/developmental toxicity screening test in rats in which dose-levels of 101, 309 and 1080 mg/kg/day elicited no toxicity.
Therefore, 1000 mg/kg/day which is also the maximum recommended dose in the OECD 414 guideline, was selected as the high-dose level. The low-dose and mid-dose have been selected using a ratio representing a three-fold interval (i.e. 100 and 300 mg/kg/day).

- Rationale for animal assignment: stratified procedure base.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice a day during the treatment period.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once a day during the treatment period.

BODY WEIGHT (GAIN):
- Time schedule: 2-3 times per week until day 21 post-coitum.

FOOD CONSUMPTION:
- Time schedule: 2-3 times per week until day 21 post-coitum.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on day 21 post-coitum.
- Examined: principal thoracic and abdominal organs
Ovaries and uterine content:
The ovaries and uterine content were examined after termination, including::
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions
- Number of uterine scars, evaluation of placenta
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: approximately half per litter
- Skeletal examinations: Yes: remaining live fetuses per litter
- Head examinations: Yes: approximately half per litter
- Other : number dead and live, body weight, sex
Statistics:
Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test.
Indices:
% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
See attached background material

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
See attached background material

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The test item was administered by gavage, once daily, from day 6 to 20 post-coitum, inclusive, to mated female Sprague-Dawley rats at dosages of 100, 300 or 1000 mg/kg/day.
Executive summary:

The objective of this prenatal developmental toxicity study was to evaluate the potential toxic effects of the test item, sodium hypophosphite, on the pregnant female and on embryonic and fetal development following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (day 6 to day 20 post‑coitum inclusive).

Methods

Three groups of 24 mated Sprague-Dawley rats were administered the test item once daily from day 6 to day 20 post-coitum, by gavage, at dosages of 100, 300 or 1000 mg/kg/day. An additional group of 24 mated females received the vehicle, purified water obtained by reverse osmosis, under the same experimental conditions and acted as the control group.A dose volume of 5 mL/kg/day was used.

 

The animals were checked daily for mortality and/or clinical signs. Body weight and food consumption were recorded at designated intervals. On day 21post-coitum (p.c.), females were sacrificed and submitted to macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.

 

Results

The test item concentrations in the administered dosage forms analyzed in weeks 1 and 3 remained within an acceptable range of variation (-5.3% to +1.0%) when compared to the nominal values.

 

Pregnancy status

At termination on day 21 p.c., there were 24, 24, 24 and 23 dams with live fetuses in the vehicle control, 100, 300 and 1000 mg/kg/day groups, respectively.

 

Mortality

There were no unscheduled deaths.

 

Clinical signs

There were no clinical observations in the 100 and 300 mg/kg/day groups. At 1000 mg/kg/day, there were a few clinical signs (piloerection and ptyalism) probably due to the taste of the test item. These findings were not considered to be adverse or toxicologically relevant.

 

Body weight, body weight change and food consumption

There were no toxicologically significant effects on mean body weights, mean body weight changes or mean food consumption.

 

Necropsy and hysterectomy data

At necropsy, there were no effects on mean gravid uterus weight and no macroscopic changes which were considered to be associated with treatment with the test item.

There were no biologically significant effects on mean hysterectomy data (pre- and post‑implantation losses and, early and late resorptions).

 

Fetal examination

There were no effects of the treatment with the test item on mean fetal body weights or fetal sex ratios.

External examination: there were no effects on the incidence of external variations per fetuses or per litter. There were no external malformations.

 

Soft tissues examination: there were no soft tissue variations and no malformations considered to be related to treatment with the test item.

 

Skeletal examination:

- There were no toxicological significant findings at examination of fetal cartilages [at 300 and 1000 mg/kg/day, the slight but statistically increases in the number of fetuses with cartilage of thoracic vertebrae present (21.0 and 25.8% vs. 12.3% in controls, p<0.05 and 0.01, respectively) were treatment-related but considered of non toxicological significance].

- At 100 and 300 mg/kg/day, there were no treatment-related variations. At 1000 mg/kg/day, there was an increased number of litters with fetuses with ossification point of the 14th thoracic vertebra (47.8% vs. 8.3% in controls, p<0.01) anda slight increase in the number of fetuses with incomplete ossification of thoracic vertebrae centrum (23.9 % vs. 11.7% in controls, p<0.01). While probably treatment-related, these findings were considered to represent slight development variations and to be non adverse.

- There were no treatment-related malformations. At 1000 mg/kg/day, an increase in the number of fetuses with absence of lumbar vertebrae was observed (1.9% vs. 0.0 % in controls). As the incidence was low, not statistically significant and in the absence of other malformations, this finding was considered to be incidental.

Conclusion

The test item was administered by gavage, once daily, from day 6 to 20 post-coitum, inclusive, to mated female Sprague-Dawley rats at dosages of 100, 300 or 1000 mg/kg/day.

 

On the basis of the results obtained in this study:

- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 1000 mg/kg/day,

- the No Observed Effect Level (NOEL) for embryo-fetal development was considered to be 300 mg/kg/day, based on increased slight development variation (increased incomplete ossifications or increased ossification points in a few fetuses at 1000 mg/kg/day),

- the NOAEL for embryo-fetal development was considered to be 1000 mg/kg/day, based on the absence of toxicologically significant effects at this dose level.

 

The test item did not elicit any teratogenic potential.