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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-07-16 till 2009-09-16
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:
other: audited draft study report
Title:
Unnamed
Year:
2010
Report Date:
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
- Analytical purity: 99.5%
- Impurities (identity and concentrations):
Phosphite (Na2HPO3): 0.14%
Calcium (Ca): 21 ppm
Iron (Fe): 0.7 ppm
Sulphate (SO4): 8 ppm
Chloride (Cl): 64 ppm
- 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 sexually mature)
- 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 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)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
DOSING:
- Dose-levels 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 concentrations prepared were 20.2, 61.8 and 215.9 mg/mL.
- Sodium hypophosphite dosage forms were prepared weekly (based on available stability data (CIT, Study N° 35771 AHS)) and stored in brown glass bottles at room temperature prior to use.

DIET PREPARATION
- Diet distributed weekly

VEHICLE:
- Vehicle used: water
- Amount of vehicle: 5 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Principle and validation of method used for chemical analysis of dosage forms:
Ion Chromatography with conductimetric detection (HPLC-IC) was used for the determination of sodium hypophosphite in dosage form samples
(method validated 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 are documented in this report.

- Determination of sodium hypophosphite concentration in dosage forms
The dosage form samples were assayed using the above 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%

- Determination of dosage form stability:
Suitability of the proposed preparation process was confirmed by analysis of the homogeneity and stability, as described in CIT/Study No. 35771 AHS. Results of this study indicated satisfactory stability of the dosage forms after 9 days storage at room temperature and no effect resulting from numerous openings of the flask containing the test item.

Duration of treatment / exposure:
Males: 15 days before mating, during the mating period (up to 3 weeks) and until sacrifice (i.e. at least 5 weeks in total)
Females: 15 days before mating, during the mating period (up to 3 weeks), during pregnancy, during lactation until day 5 post-partum inclusive.
(day 1 = first day of treatment period)
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
101, 309, 1080 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose-levels were selected on the basis of a 2-week dose Range-Finding toxicity study (CIT, Study N° 35768 TSR) reported in chapter 7.5.1

Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- From arrival: once a day as part of routine examinations
- From the start of the treatment: once a day, at approximately the same time for the recording of clinical signs

MORBIDITY/MORTALITY:
Once a day during the acclimation period and at least twice a day during the treatment period

DETAILED CLINICAL OBSERVATIONS:
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:
- 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 (FC):
- Males: recorded weekly over a 7 day period from the first day of treatment until sacrifice.
- Females: recorded 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.

HAEMATOLOGY:
- Time schedule for collection of blood: On the day of sacrifice after food deprivation for an overnight period of at least 14 hours
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: first 5 males and first 5 females to deliver from each group
- Parameters checked in table 1 were examined

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: on the day of sacrifice
- How many animals:first 5 males and first 5 females to deliver from each group
- Parameters checked in table 1 were examined

URINALYSIS:
- Time schedule for collection of urine: on the day of sacrifice after food deprivation for an overnight period of at least 14 hours
- Animals fasted: Yes
- How many animals: first 5 males and first 5 females to deliver from each group
- Parameters checked in table 1 were examined

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule for examinations: first 5 males and first 5 females to deliver from each group were evaluated at the end of the treatment period (for females, on day 5 post-partum after sacrifice of the pups)
- Dose groups that were examined: all, animals were randomized to ensure "blind" evaluation
- Observation: in the cage, in the hand and in the standard arena
- 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)
- Parameters assessed and graded and parameter measurements, reflexes and responses recorded afterwards are given in table 2
Sacrifice and pathology:
SACRIFICE
males: after the end of the mating period (after at least 5 weeks of treatment)
females: on day 6 post-partum, or when not delivered by day 25 post-coitum sacrificed on this day (after body weight recording to check for a possible unnoticed delivery)

ORGAN WEIGHTS
- Organs listed in table 3 were weighted

GROSS PATHOLOGY / HISTOPATHOLOGY
1. Microscopic examination
* Preservation of tissues
The tissues listed in table 3 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.

* Microscopic examination was performed on:
All macroscopic lesions
All animals found dead
All tissues listed in table 3 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)

2. A complete macroscopic post-mortem examination was performed on all animals, also those found dead.
Other examinations:
No data
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 blood biochemistry
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)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
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
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
- Clinical signs:
No relevant clinical signs were observed during the study
- Mortality:
One female treated at 101 mg/kg/day was found dead after treatment on day 5 post-coitum, but the death was considered accidental. This female had jumped out of the weighing container before treatment and although alive at treatment, was found dead a short while later. At necropsy, a red
content in the thoracic cavity and an irregular red color of lungs which correlated histologically with hemorrhage and edema were observed. Hemorrhage was also observed in the heart and the mediastinum. These findings are probably related to injury sustained during the fall.

BODY WEIGHT AND WEIGHT GAIN (see table 1 in remarks on results)
There were no treatment-related effects.

FOOD CONSUMPTION AND COMPOUND INTAKE (see table 2 in remarks on results)
There were no treatment-related effects.

HAEMATOLOGY
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 3 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 4 in remarks on results) but the incidence and the protein level were not dose-related. 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.

NEUROBEHAVIOUR
- Functional Observation Battery (abnormal responses/observations) ans motor activity:
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 presented in table 5 (see remarks on results)

ORGAN WEIGHTS
- Thymus (see table 6 in remarks on results)
There was a lower absolute and relative weight of the thymus in females given 309 or 1080 mg/kg/day, reaching a statistically significant level at
1080 mg/kg/day. This was not associated with any histopathological change at 1080 mg/kg/day.
- Kidneys
The absolute and relative weights of kidneys were greater in females given 1080 mg/kg/da, reaching a statistically significant level 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
No macroscopic changes were noted that were considered to be associated with treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
- Forestomach:
Minimal thickening of the limiting ridge of the forestomach associated in most cases with minimal increased exocytosis of inflammatory cells, 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 was considered to be related to treatment with the test item. However, due to the minimal degree of severity of this change compared with the dose-level (more than 1000 mg/kg/day), this
effect was considered to be non-adverse and in the absence of a similar structure in the stomach from human beings, this change was not
considered 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 andbiochemical 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

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 080 mg/kg bw/day (actual dose received)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

- No contaminants were present in the diet, drinking water, wood shavings or sawdust at levels which could be expected to interfere with, or prejudice, the outcome of the study.

 

- 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) and body weight gain (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

/

/

/

/

Days 1-15

+56

+55

+56

+53

+18

+12

+17

+19

Days 1-36

+103

+105

+115

+107

/

/

/

/

Gestation

Day 0 p.c.

/

/

/

/

283

276

275

283

Day 20 p.c.

/

/

/

/

427

418

416

440

Days 0-20 p.c.

/

/

/

/

+144

+142

+142

+157

Lactation

Day 1 p.p.

/

/

/

/

325

327

312

332

Day 5 p.p.

/

/

/

/

345

354

333

354

Days 1-5 p.p.

/

/

/

/

+20

+27

+21

+22

/: 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: 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 4: 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 5: Neurobehaviour. Abnormal responses/observations noted at the end of the treatment of male (M) and female (F) rats of 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

 

 

 

- Table 6: 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)

Applicant's summary and conclusion

Conclusions:
Under these experimental conditions, no noteworthy toxic effects were observed related to the repeated oral administration of the test item sodium hypophosphite.
Executive summary:

The potential toxicity of sodium hypophosphite was evaluated following repeated oral administration to rats according to OECD Guideline 422 and EPA guideline OPPTS 870.3650. The study was conducted in compliance with the principles of Good Laboratory Practice regulations on the monohydrated form of the test item as the anhydrous form is highly hygroscopic and difficult to handle without specific precautions.

Three groups of 10 males and 10 females were administered the test substance once daily by gavage for 2 weeks before mating, during mating (up to 3 weeks), and, for the females, through gestation until day 5 post-partum at dose-levels of 101, 309 and 1080 mg/kg/day. A concurrent vehicle control group received purified water.

Clinical signs and mortality were checked daily 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.

Males were sacrificed after completion of the mating period and females on day 6 post-partum. Afterwards the designated organs were weighed and gross pathology as well as histopathology were performed.

There were no treatment-related unscheduled deaths during the study and no relevant clinical signs were observed. There were no treatment-related effects on body weight, body weight gain or food consumption at any dose-level. The functional observation battery assessment and hematology and blood biochemistry revealed no treatment-related effects but males from all groups had protein in the urine at a non-dose-related incidence.

There were no treatment-related macroscopic findings and the only effect on organ weights was a lower thymus weight in females treated at 309 or 1080 mg/kg/day which, in the absence of histopathological changes was considered to be of low toxicological significance. Microscopic examination revealed minimal increased epithelial thickness of the forestomach, in males treated at 309 mg/kg/day and in both sexes treated at 1080 mg/kg/day.

This change was considered to be related to treatment with the test item. However, due to the minimal degree of severity of this change compared with the administered dose-level (more than 1000 mg/kg/day), this effect was considered to be non-adverse and in the absence of a similar structure in the stomach from human beings, this change was not considered relevant for human health.

Under these experimental conditions the No Observed Adverse Effect Level (NOAEL) is 1080 mg/kg/day.