Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-07-16 till
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study (OECD 422 Guideline, EPA OPPTS 870.3650 Guideline)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA Guideline OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material: Sodium hypophosphite; tested in the form of monohydrate as the anhydrous form (CAS 7681-53-0) is highly hygroscopic and difficult to handle without specific precautions.
- Physical state: white crystalline solid
- Supplier: Rhodia UK-Oldbury
- Batch number: 90113D
- Expiry date: June 2010
- Storage condition of test material: room temperature, stored in a tightly closed container in a dry and well-ventilated place, opening of the container restricted to the minimum needs
- Disposal: incinerate the residues at an approved waste disposal site only
- Purity: 99.5%
- Impurities (identity and concentrations):
Phosphite (Na2HPO3): 0.14%
- Certificate of analysis:
Analysis number: ITC/09/01/04
Analysis date: 2009-01-27

No more data available

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France (l'Arbresle, France)
- Strain and Sanitary status: Sprague-Dawley Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®)
- Age at study initiation: males approx. 12 weeks old and females approx. 11 weeks old. Both sexes were sexually mature and females were primigravid
- Weight at study initiation:
males: mean body weight = 394 g (range: 353 g to 436 g)
females: mean body weight = 258 g (range: 238 g to 282 g)
- Identification: individual ear tattoo (unique CIT identity number)
- Housing: individually, except during pairing, in wire mesh cages; females were housed in polycarbonate cages towards the end of the gestation period and with their litter during lactation. Autoclaved wood shavings were provided as nesting material, a few days before delivery and during the lactation period
- Diet: ad libitum - SSNIFF R/M-H pelleted maintenance diet, batch No. 9361978 and 8632516 (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: ad libitum - 0.22 µm filtered tap water
- Acclimation period: 5 days, one supplementary animal of each sex was acclimated to permit the selection and/or replacement of individuals if necessary
- Contaminant analyses:
batches of diet, wood shavings and sawdust analyzed by suppliers for composition and contaminant levels
bacterial and chemical analyses of water regularly by external laboratories (analyses included detection of possible contaminants such as pesticides, heavy metals and nitrosamines)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approx. 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12/12 (7:00 - 19:00)

No more data available

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dose-levels were calculated in terms of sodium hypophosphite monohydrate (CAS 10039-56-2, MW 103.977).
The test item was mixed with the required quantity of vehicle and the concentrations prepared were 20.2, 61.8 and 215.9 mg/mL.
The sodium hypophosphite dosage forms were prepared weekly (based on available stability data (CIT, Study N° 35771 AHS)) and were stored in brown glass bottles at room temperature prior to use.

DOSING:
- Quantity of dosage: adjusted according to the most recently recorded body weight of each animal
- Constant dosage-volume: 5 mL/kg/day
- Dosage forms: magnetically stirred continuously throughout the dosing procedure

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

VEHICLE
- Amount of vehicle: 5 mL/kg/day

No more data available
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: females were placed with males from the same dose-level group during the night; females were placed with the same male until mating occurred or 14 days had elapsed.
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage referred to as day 0 post-coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Principle and validation of the method used for the chemical analysis of the dosage forms
The Ion Chromatography with conductimetric detection (HPLC-IC) method was used for the determination of sodium hypophosphite in dosage form samples (method validated at CIT prior to dosage form analysis). Validation of the method was based on the ICH Q2(R1) guideline adopted in October 1994 and accordingly the following parameters were checked: specificity, precision and accuracy, repeatability, linearity, Sensitivity Evaluation Test (SET) and stability of the test item in working solutions.
The validation of the analytical method was conducted in CIT/Study No. 35770 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results are documented in the validation report.

Composition of an analytical sequence
Each analytical sequence consisted of at least: a blank sample (dilution solvent), ten standard samples at nominal concentration, prepared from two independent solutions and study samples prepared from aliquots of the dosage forms. The standard samples bracketed the dosage form samples. The blank sample checked for the absence of chromatographic interferences.

Analytical sequence suitability rules
Acceptance criteria are defined in CIT Standard Operating Procedures (SOPs). These acceptance criteria include (1) precision of the standard solutions [%RSD (Relative Standard Deviation) must pass defined acceptance criteria], and (2) agreement of the standards (the mean response factor of standard samples prepared from solution #1 must agree with the mean response factor of standard samples prepared from solution #2).

Determination of dosage form stability
The suitability of the proposed preparation process was confirmed by analysis of the homogeneity and stability, as described in CIT/Study No. 35771 AHS. In this study a range of dosage forms was prepared at levels which covered the lowest and highest concentrations proposed for use in the study, and then stored at room temperature.
Results indicated satisfactory stability of the dosage forms after 9 days storage at room temperature and also not affect as a result of numerous openings of the flask containing the test substance.

Determination of sodium hypophosphite concentration in dosage forms
The dosage form samples were assayed using a validated method. The test item concentration in samples of each control and test item dosage form prepared for use in weeks 1, 4 and 7 was determined.
Acceptance criterion was measured concentration = nominal concentration ± 10%
Duration of treatment / exposure:
Males: premating period (15 days) + mating period (up to 3 weeks); until sacrifice (i.e. at least 5 weeks in total)
Females: premating period (15 days) + mating period (up to 3 weeks) + gestation + lactation (until day 5 post-partum inclusive)
Frequency of treatment:
Once daily at approximately the same time
Details on study schedule:
No additional data available
Doses / concentrations
Remarks:
Doses / Concentrations:
101, 309, 1080 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10 Males & 10 Females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: selection based on a preliminary 2-week toxicity study in the rat (CIT, Study N° 35768 TSR). In this preliminary study the dose-levels were 101, 309 and 1080 mg/kg/day and none elicited any toxicity. Hence, the same dose-levels were selected for this study.

- Rationale for animal assignment: during acclimation period, the required number of animals were selected according to body weight and clinical condition. Animals were allocated to groups (by sex), according to a computerized stratification procedure so that the average body weight of each group was similar.

No more data available
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- From arrival till start of treatment: once a day as part of routine examinations
- From the start of the treatment: once a day, at approximately the same time for recording of clinical signs
- Mortality and morbidity: once a day during the acclimation period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS: Yes
Once before the beginning of the treatment period and then once a week until the end of the study.
Detailed clinical observations were performed outside the home cage in a standard arena. Observations included (but were not limited to):
- Changes in the skin, fur, eyes and mucous membranes
- Occurrence of secretions and excretions
- Autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern)
- Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded

BODY WEIGHT: Yes
- Males: on the first day of treatment (day 1), then weekly until sacrifice
- Females: on the first day of treatment (day 1), then weekly 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
- Males: determined weekly over a 7 day period from the first day of treatment until sacrifice.
- Females: determined weekly over a 7 day period from the first day of treatment through gestation (days 0-7, 7-14 and 14-20 post-coitum intervals) and lactation (days 1-5 post-partum intervals) until sacrifice.
- During the pairing period, food consumption was not recorded for males or females.

OTHER:
LABORATORY PARAMETERS - HAEMATOLOGY/CLINICAL CHEMISTRY/URINALYSIS (F0 generation) (see table 1 in Any other information on materials and methods for parameters studied)
- Haematology
Blood was sampled for the first 5 males and the first 5 females to deliver from each group on the day of sacrifice after they were deprived of food for an overnight period of at least 14 hours. Light anaesthetic used for blood collection from the orbital sinus was isoflurane
- Clinical chemistry
Blood was collected on the day of sacrifice for the first 5 males and the first 5 females to deliver from each group
- Urinalysis
Urine was collected on the day of sacrifice for the first 5 males and the first 5 females to deliver from each group after they were deprived of food for an overnight period of at least 14 hours

NEUROBEHAVIOURAL EXAMINATION (F0 generation)
- Time schedule for examinations: the first 5 males and the first 5 females to deliver from each group were evaluated at the end of the treatment period (for females, this was on day 5 post-partum after sacrifice of the pups)
- Dose groups that were examined: all, animals were randomized in order to ensure "blind" evaluation
- Battery of functions tested:
    Detailed clinical examination
    Measurement of reactivity to manipulation
    Measurement of reactivity to different stimuli
    Motor activity (measured once by automated infra-red sensor equipment over a 60-minute period)
- Observation: in the cage, in the hand and in the standard arena
- Parameters assessed and graded:
    "touch escape" or ease of removal from the cage
    In the hand
         fur appearance
         salivation
         lachrymation
         piloerection
         exophthalmos
         reactivity to handling
         pupil size (presence of myosis or mydriasis)
    In the standard arena (2-minute recording)
         Grooming
         palpebral closure
         defecation
         urination
         tremors
         twitches
         tonic and clonic convulsions
         gait
         arousal (hypo- and hyper-activity)
         posture
         stereotypy
         behavior
         breathing
         ataxia
         hypotonia
- Parameters measured, reflexes and responses recorded:
    touch response
    forelimb grip strength
    pupillary reflex
    visual stimulus response
    auditory startle reflex
    tail pinch response
    righting reflex
    landing foot splay
    rectal temperature (at the end of observation)
- Motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period
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 had mated
Sperm parameters (parental animals):
Parameters examined in male parental generation (F0):
epididymis and testis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups,
- postnatal mortality and cannibalized pups,
- presence of gross malformations,
- clinical signs,
- body weight (recorded on days 1 and 5 post-partum)

GROSS EXAMINATION OF DEAD PUPS: examination for gross external abnormalities and a macroscopic examination. No preservation of tissues
Postmortem examinations (parental animals):
SACRIFICE
- After overnight fasting, at least 14 hours (except for females sacrificed during the gestation period), all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination. Exception were females sacrificed during the gestation period
Males: after the end of the mating period (after at least 5 weeks of treatment)
Females: on day 6 post-partum, or on day 25 post-coitum for females that had not delivered (after body weight recording to check for a possible unnoticed delivery)

GROSS NECROPSY
- Complete macroscopic post-mortem examination was performed on all animals, also those found dead. This included: the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.
- Special attention was paid to the reproductive organs. Numbers of corpora lutea and implantation sites were recorded for females sacrificed on day 6 post-partum as well as for females sacrificed on day 25 post coitum due to no delivery. Implants were classified as uterine scars, early or late resorptions or live or dead concepti. For apparently non-pregnant females the presence of implantation scars on the uterus was also checked using the ammonium sulphide staining technique

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathology
* Preservation of tissues
The tissues cited in table 2 (see Any other information on materials and methods) were preserved for the first 5 sacrificed-as-scheduled males, the first 5 females to deliver and be sacrificed on day 6 post-partum of each group, and for animals found dead unless otherwise specified. The following tissues are an exception and were preserved for all animals: epididymides, ovaries (with oviducts), prostate, seminal vesicles, testes, uterus (horns and cervix) and vagina. Preservation of all the tissues was done in 10% buffered formalin (except for the testes and epididymides which were preserved in Davidson’s fixative).
* Examination was performed on:
All macroscopic lesions
All animals found dead
All tissues listed in table 2 (see Any other information on materials and methods) for the first 5 sacrificed-as-scheduled males and the first 5 females to deliver and be sacrificed on day 6 post-partum of the control- and high-dose groups (1080 mg/kg/day).
The stomach with forestomach and the liver of the first 5 sacrificed-as-scheduled males and the first 5 females to deliver and be sacrificed on day 6 post-partum of the intermediate dose group (309 mg/kg/day)
Special emphasis was on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure
- Organ weights (organs weighed are listed in table 2 in the section Any other information on materials and methods)
Body weight of each animal was recorded before sacrifice at the end of the treatment period.
In general the organ weights were determined for the first 5 sacrificed-as-scheduled males and the first 5 females to deliver and be sacrificed on day 6 post-partum of each group. The organs were weighed wet as soon as possible after dissection .The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.
Postmortem examinations (offspring):
- Surviving pups were sacrificed on day 5 post-partum, without fasting, by an intraperitoneal injection of sodium pentobarbital
- These animals were carefully examined externally for gross external abnormalities and a macroscopic examination was performed. There was no preservation of tissues.
Statistics:
- Body weights, food consumption and reproductive data
Mean values were compared by one-way variance analysis and Dunnett test (mean values being considered as normally distributed, variances being considered as homogeneous).
Percentage values were compared by Fisher exact probability test.

- Hematology and clinical chemistry
Citox software (version d.5) was used to perform the statistical analysis of hematology, blood biochemistry and urinalysis data

- Organ weights
PathData software (version 6.2b5) was used for the statistical analysis of organ weight data (level of significance: 0.05 or 0.01)
Reproductive indices:
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 5 post-partum = (Number of surviving pups on day 5 post-partum/Number of live born pups) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
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
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Clinical signs:
Animals did not exhibit relevant clinical signs during the study
- Mortality:
One unscheduled death was observed. A female treated at 101 mg/kg/day was found dead after treatment on day 5 post-coitum. This female had jumped out of the weighing container before treatment and although she was alive at treatment, a short while later was found dead. At necropsy, there was a red content in the thoracic cavity and an irregular red color of lungs which correlated histologically with hemorrhage and edema. Hemorrhage was also observed in the heart and the mediastinum. Hence, the death was considered accidental and probably related to injury sustained during the fall.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight (see table 1 in remarks on results)
Males: no effects on mean body weight at any dose-level throughout the study
Females: no effects on mean body weight during the pre-mating, gestation or lactation phase. All values were comparable with the control group.
- Food consumption (see table 2 in remarks on results)
No effects on mean male or female food consumption throughout the study

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
One control female stayed in diestrus for 15 days but the female mated on the 16th day and was pregnant. No other females showed abnormal estrous cycling.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Mating (see table 3 in remarks on results)
There were no effects of treatment with the test item on mating; only one control pair did not mate and the mean number of days of pairing before mating was less than the controls at all dose-levels.
- Fertility (see table 3 in remarks on results)
Two females treated at 101 mg/kg/day and one female in each of the groups treated at 309 or 1080 mg/kg/day mated but were not pregnant. This rate of pregnancy fell within the historical data range for this species and no dose-relationship was observed, therefore this is considered to be fortuitous
- Delivery (see table 4 in remarks on results)
No effects noted on the mean numbers of corpora lutea, implantations or pups at any dose level, nor on the duration of gestation or the extent of pre- and post-implantation loss.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Thymus (see table 5 in remarks on results)
Significant differences (P<0.05) were observed for absolute and relative weight of the thymus between the females of the control group and the females dosed at the highest concentration (1080 mg/kg/day). There were no histological changes which correlated with the lower weight of the thymus in females dosed at 1080 mg/kg/day.
The lower non-statistically significant weight of the thymus noted in females given 309 mg/kg/day was considered to be probably associated with the test item but of low toxicological significance.
- Kidneys
Although the absolute and relative weights of kidneys were greater in females given 1080 mg/kg/day than in the female control group, this difference was only statistically significant for the relative weight (P<0.05). In the absence of any treatment-related changes in biochemistry or urinary parameters and at the histopathological examination, this variation was considered to be incidental.
- Other changes in the mean organ weights were considered to reflect normal individual variations and were considered to be unrelated to treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No macroscopic changes were noted that were considered to be associated with treatment. The observed changes were considered to be part of the normal background in the rat

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Forestomach:
minimal thickening of the limiting ridge was observed in 5/5 males and 3/5 females given 1080 mg/kg/day and in one male given 309 mg/kg/day. This change was characterized by minimal increase in epithelial thickness and more prominent rete pegs. In most animals given 1080 mg/kg/day, it was associated with minimal increased exocytosis of inflammatory cells (mainly mononuclear cells).
Considering the minimal degree of severity of this change compared with the dose-level (more than 1000 mg/kg/day), this change can be considered to be non adverse. Moreover, since a similar structure in the stomach from human beings is absent, the observation is considered not relevant for human health
- Liver:
In the liver of females, minimal foci of hepatocellular necrosis were observed in the subcapsular area of 1/5 females given 309 mg/kg/day and 3/5 females given 1080 mg/kg/day. This change characterized by the presence of aggregates of macrophages, occasionally with pigment and some fibroblasts, was not acute and was considered to reflect the healing of previous necrotic areas. Subcapsular hepatocellular necrosis may be occasionally seen in rats of this strain and age. Therefore, despite the apparent dose-related increased incidence, this observation was considered to be probably incidental, because of its low magnitude, the location restricted to the subcapsular area as well as the absence of change in males, in liver weights and biochemical parameters.
- Thymus:
At necropsy no histopathological changes which correlated with the lower weight of the thymus were noted
- Testes:
Careful examination did not reveal any treatment-related changes

OTHER FINDINGS (PARENTAL ANIMALS)
LABORATORY PARAMETERS - HAEMATOLOGY/CLINICAL CHEMISTRY/URINALYSIS
- Haematyology:
No remarkable differences between control and test item-treated animals
- Clinical chemistry:
A statistically significant decrease in creatinine in females treated at 1080 mg/kg/day was observed (see table 6 in remarks on results). This was considered incidental as a similar pattern was not observed in males and no concomittant parameters were affected.
- Urinalysis:
Protein levels: All test item-treated male groups had higher levels of protein in the urine than the controls (see table 7 in remarks on results). However, no clear dose-relationship was observed and no female showed protein in the urine so the significance of this observation is unclear.
Blood: One male treated at 101 mg/kg/day had blood in the urine but the isolated nature of this finding indicates that it is not related to treatment with the test item.

NEUROBEHAVIOURAL EXAMINATION
- Functional Observation Battery (abnormal responses/observations):
No dose-relationship was observed in the abnormal responses and often one animal was concerned in several tests. As no relevant clinical signs were noted during the study, these abnormal responses were considered not indicative of an effect of treatment with the test item. The abnormal responses/observations noted during the study are shown in table 8 (see remarks on results)
- Motor activity
No differences in the mean number of movements made by the animals of all groups when compared with the controls

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 080 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEL
Effect level:
1 080 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: reproductive performance (mating and fertility)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Pup survival was lower in the control group than in the groups treated with sodium hypophosphite.

CLINICAL SIGNS (OFFSPRING)
Isolated clinical signs were observed in a few animals per test item-treated group, but no one sign was observed consistently and the control pups had the most clinical signs. Thus, it was considered that there were no effects of treatment with the test item.

BODY WEIGHT (OFFSPRING)
No effects of treatment with the test item were observed on the mean pup body weight of any group

GROSS PATHOLOGY (OFFSPRING)
One pup from the group treated at 1080 mg/kg/day had pale kidneys. Hemorrhagic left testis and dilated renal pelvis were each recorded in one control pup. Considering the low incidence of all these findings, these observations do not indicate a relationship to the treatment .

OTHER FINDINGS (OFFSPRING)
SEX-RATIO
The percentage of male pups was statistically significantly higher at 101 mg/kg/day than in the control group although no differences were observed at 309 or 1080 mg/kg/day. Hence, this observation was considered to be incidental.

Effect levels (F1)

Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 080 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: toxic effects on progeny

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

- Chemical analysis of the dosage forms

Test item concentrations in the administered dosage forms were analyzed in weeks 1, 4 and 7 of treatment and remained within an acceptable range of -0.8% to +3.0% of variation compared to the nominal values.

- Table 1: Mean body weight (g) for male and female rats of all treatment groups are presented in the table below

Sex

Male

Female

Dose-level (mg/kg/day)

0

101

309

1080

0

101

309

1080

Pre-mating

Day 1

396

394

393

392

257

260

255

259

Day 15

452

448

448

445

275

272

272

278

Day 36

499

499

507

499

/

/

/

/

Gestation

Day 0 p.c.

/

/

/

/

283

276

275

283

Day 20 p.c.

/

/

/

/

427

418

416

440

Lactation

Day 1 p.p.

/

/

/

/

325

327

312

332

Day 5 p.p.

/

/

/

/

345

354

333

354

/: not recorded, p.c.:post-coitum, p.p.: post-partum

- Table 2: Mean food consumption (g/day) for male and female rats of all treatment groups are presented in the table below

Sex

Male

Female

Dose-level (mg/kg/day)

0

101

309

1080

0

101

309

1080

Pre-mating

Day 1-8

34

32

31

31

20

21

20

20

Days 8-15

35

33

32

32

22

21

22

21

Gestation

Day 0 -7 p.c.

/

/

/

/

26

25

27

27

Days 7-14 p.c.

/

/

/

/

28

27

28

29

Days 14-20 p.c.

/

/

/

/

31

30

30

32

Lactation

Days 1-5 p.p.

/

/

/

/

43

48

44

46

/: not recorded, p.c.: post-coitum, p.p.: post-partum

-Table 3: Summary of mating and fertility data

Dose-level (mg/kg/day)

0

101

309

1080

Mating

Males

Males paired (N)

10

10

10

10

Males mated (N)

9

10

10

10

Males mating index (%)

90

100

100

100

Females

Females paired (N)

10

10

10

10

Females mated (N)

10

10

10

10

Females mating index (%)

100

100

100

100

Mean number of days of pairing before mating

4.0

2.1

2.1

2.7

Fertility

Pregnant female partners (N)

10

8

9

9

Fertility index (%)

100

80

90

90

Females with live concepti (N)

10

8

9

9

Gestation index (%)

100

100

100

100

-Table 4: Summary of delivery data

Dose-level (mg/kg/day)

0

101

309

1080

Number of pregnant females surviving delivery

10

8

9

9

Mean duration of gestation (days)

21.0

21.3

21.2

21.2

Mean number ofcorpora lutea

19.2

15.9

16.6

18.1

Mean number of implantations

15.2

13.8

15.7

16.4

Mean pre-implantation loss (%)

18.7

13.0

5.1

7.4

Number of litters with liveborn pups

10

8

9

9

Mean number of pups delivered

14.4

13.1

15.1

16.0

Mean post-implantation loss (%) (calculated manually)

7.6

4.8

3.6

3.0

 

-Table 5: Organ weights. The differences between the control and the treated group (expressed in %) are shown for body weight and absolute and relative weight of the thymus.

Sex

Male

Female

Dose-level (mg/kg/day)

101

309

1080

101

309

1080

Body weight

-2%

+2%

+1%

+3%

-4%

+1%

Thymus

absolute

+3%

+4%

+4%

-7%

-27%

-36% *

relative

+7%

+1%

+2%

-7%

-22%

-36% *

* p<0.05 (Organ weight value statistically significant from control; not the percentage)

- Table 6: Clinical chemistry. Overview of creatine concentrations in blood for all treatment groups for both sexes

Sex

Male

Female

Dose-level (mg/kg/day)

0

101

309

1080

0

101

309

1080

Creatinine (μmol/L)

46

50

50

49

49

49

47

45*

Statistically significant, *: p<0.05

 

- Table 7: Urinalysis. Occurence and levels of proteins for both sexes and all treatment groups are presented as well as the occurence of blood in urine.

Sex

Male

Female

Dose-level (mg/kg/day)

0

101

309

1080

0

101

309

1080

Protein

Negative

4/5

2/5

 

2/5

5/5

5/5

5/5

5/5

0.3 g/L

1/5

2/5

4/5

2/5

 

 

 

 

1 g/L

 

 

1/5

1/5

 

 

 

 

3 g/L

 

1/5

 

 

 

 

 

 

Blood

negative

 5/5

4/5

5/5 

5/5 

 5/5

 5/5

5/5 

 5/5

  moderate

 

 1/5

 

 

 

 

 

 

- Table 8: Neurobehaviour. Abnormal responses/observations noted at the end of the treatment of male (M) and female (F) rats for all treatment groups

Dose-level (mg/kg/day)

0

101

309

1080

Difficulty in removal from cage

 

1M

 

 

Difficulty in handling

 

1M

 

 

Hypoactivity

 

 

 

1F

Bizarre response to touching; aggressiveness, vocalization or biting

1M

1M

 

 

Bizarre response to touching; freezing or withdrawal

 

 

 

1M

Freezing or withdrawal during visual stimulus

 

2M

 

 

No response to tail pinch

1M

 

 

1M

Hyperactivity, aggressiveness or biting at tail pinch

1M

1M

1M

1M

Slight difficulty during righting reflex test

1M, 1F

1M

1F

 

Fall on back during righting reflex test

 

 

1M

 

Moderate decrease in forelimb grip strength

1M

1M

1M

2M

Increase in forelimb grip strength

2M

1M

1F

2M, 1F

No forelimb grip

 

1M

 

 

Applicant's summary and conclusion

Conclusions:
Under these experimental conditions, the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 1080 mg/kg/d. The NOEL for reproductive performance (mating and fertility) is 1080 mg/kg/day and the NOEL for toxic effects on progeny is 1080 mg/kg/day.
Executive summary:

The potential toxicity of sodium hypophosphite was evaluated according to OECD Guideline 422 (Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test). Possible toxicological effects likely to arise from repeated exposure on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition were investigated.

Sodium hypophosphite was administered orally once a day by gavage to Sprague-Dawley rats (10 males and 10 females per treatment group) at dose-levels of 101, 309 and 1080 mg/kg/d for 2 weeks pre-mating, during mating (up to 3 weeks), and for the females through gestation until day 5post-partum. A control group was included (10 males and 10 females) which was kept under the same conditions as the exposed animals and received only the vehicle, which was purified water obtained by reverse osmosis. The dosing volume was 5 mL/kg/day.

Clinical signs and mortality were checked once and twice a day respectively and detailed clinical observations were performed weekly. Body weight and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. A Functional Observation Battery including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditary startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed at the end of the study. Blood and urine samples were taken for analysis of haematology, clinical chemistry and urinalysis at the end of the study. After 15 days of dosing, the females were paired with males from the same dose-level group for mating and gestation was monitered. The females 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. Pup clinical signs were recorded daily and pup body weights were recorded on days 1 and 5 post‑partum.

Males were sacrificed after completion of the mating period and the females on day 6 post-partum. A complete macroscopic examination was performed with particular attention to the reproductive organs. Afterwards the designated organs were weighed and gross pathology as well as histopathology were performed.

Pups sacrificed on day 6 post-partum and those found dead, were carefully examined for gross external abnormalities and a macroscopic post-mortem examination was performed.

There were no treatment-related unscheduled deaths during the study and no relevant clinical signs were observed. There were also no treatment-related effects on body weight or food consumption at any dose-level. The functional observation battery and motor activity assessment did not show any treatment related effects. Haematology and cli nical chemistry revealed no treatment-related effects, while urinalysis showed that males from the exposed groups had protein in the urine in a non-dose-related manner. There were no differences from controls for mating and fertility parameters. Also the mean number of corpora lutea, implantations and the mean number of live born pups were all comparable to the controls. The test item did not exert any effects on pup development in utero an also no effects of treatment on pup survival, clinical signs, sex ratio or body weight performance after birth. There were no treatment-related macroscopic findings and the only observed effect on organ weights was a lower absolute and relative thymus weight in females treated at 309 or 1080 mg/kg/day. For the latter dose level this effect was statistically significant (p<0.05). However this effect could not be associated with any histopathological change. Finally, microscopic examination revealed minimal increased epithelial thickness of the forestomach in males treated at 309 mg/kg/day (1/5 males) and in both sexes treated at 1080 mg/kg/day (5/5 males and 3/5 females). Although this effects could be related to treatment it was considered non-adverse because of the limited degree of severity. Furthermore, as a similar structure is absent in the stomach of humans, the effect is not considered relevant for human health. In the liver, minimal foci of hepatocellular necrosis were observed in 1/5 females given 309 mg/kg/day and 3/5 females given 1080 mg/kg/day. This change was observed only in females and was not associated with liver weight or histopathological changes. As such changes may be seen in rats of this strain and age, they are considered incidental.

Consequently, under the experimental conditions the No Observed Adverse Effect Level (NOAEL) for parental toxicity is 1080 mg/kg/d. The NOEL for reproductive performance (mating and fertility) is 1080 mg/kg/day and the NOEL for toxic effects on progeny is 1080 mg/kg/day.

This study is considered as reliable without restrictions as it is a GLP certified guideline study (OECD 422). Furthermore, according to regulation 67 -548 EEC and CLP regulation (EC) N° 1272/2008 sodium hypophosphite is not classified as a reproductive toxicant and also does not exhibit effects on or via lactation.