Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002-05-28 - 2002-12-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to
Guideline:
OECD Guideline 424 (Neurotoxicity Study in Rodents)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
- Synonyms: ITC 908, vinylidenephosphonic acid tetrasodium salt (VDPA tetrasodium salt)
- Lot/batch No.of test material: 562CH/646
- Purity: 87.33%
- Storage condition of test material: Room temperature, in the dark

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle France
- Age at study initiation: 6 weeks old
- Weight at study initiation: 204g for males and 169g for females
- Fasting period before study:
- Housing: Suspended wire-mesh cages. A metal tray containing autoclaved sawdust was placed under each cage.
- Diet: A04 C pelleted maintenance diet (UAR, Villemoisson, Epinay-sur-Orge, France), free access out of fasting periods
- Water: ad libitum
- Acclimation period: 8 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- Humidity (%): 50 +- 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
By gavage using a plastic syringe fitted with a metal gavage tube, once a day, at approximately the same time.
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was mixed with the required quantity of water in order to achieve the concentrations of 10, 30 and 120 mg/ml and then homogenized using a magnetic stirrer. The test item dosage forms were prepared on a weekly basis and stored at +4 °C prior to use.

VEHICLE
- Concentration in vehicle: 10, 30 and 120 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:
Before the start of the treatment, the suitability of the proposed dosage form formulation procedure was determined. During the treatment period, the concentration of dosage forms was checked (concentration in the samples prepared for use in weeks 1 and 4).
Dosage forms at the lowest and highest concentrations proposed for use in the study (i.e. 10 and 120 mg/ml) were tested for stability.

The results of the analyses demonstrated the satisfactory stability of the investigated dosage forms (10 mg/ml and 120 mg/ml) over a 9-day period at +4 °C. Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test item in the administered dosage forms since the deviations from nominal concentration were in an acceptable range of +- 8%.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 7-day range-finding toxicity study
Poor clinical conditions, severly lower body weight gain and food consumption and changes in organ weights and macroscopic examination were noted in animals given 1000 mg/kg.

- Fasting period before blood sampling for clinical biochemistry: 14 hours

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before the beginning of the treatment period and then once a week

BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation to a group, on days 1, 8, 15, 22 and 28.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28 before the gavage treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28
- Animals fasted: Yes
- How many animals: All

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 28
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the treatment period, after at least 14 hours fasting, all animals were asphyxiated by carbon dioxide and killed by exsanguination.

Observations
* Organ weights
* Macroscopic post-mortem examination
* Microscopic examination
Statistics:
The results were analyzed using a Dunn test, a Mann-Whitney/Wilcoxon test, a Dunnett test or a Student test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed in 4/5 males and 5/5 females given 600 mg/kg/day (generally from day 8 or 22) and in 1/5 males given 150 mg/kg/day (from day 22). This sign, which is commonly noted with test items administered by gavage, was not considered as an adverse effect. In addition, signs of poor clinical condition (piloerection and round back) were observed in 1/5 females given 600 mg/kg/day from day 27).

All other clinical signs, namely area of hair loss, cutaneous lesions and scabs (observed in 1/5 males given 50 mg/kg/day) were not dose-related, not observed with the same trend in both sexes and are commonly seen in the laboratory rat of this strain and age. They were thus not considered to be related to treatment with the test item.

Abnormal quantity or aspect of feces was observed in 1/5 control males and 1/5 males given 600 mg/kg/day; as it was observed in both control and treated animals and without the same trend in the two sexes, a relationship to treatment is ruled out.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Slightly lower mean body weight gain was observed in females given 150 and 600 mg/kg/day (decreasing trend).
However, no significant effects were noted for males.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
When compared to controls, no relevant differences in the mean food consumption were noted in any treated animals.
Haematological findings:
no effects observed
Description (incidence and severity):
No relevant differences of toxicological importance were noted between control and treated animals. The only differences observed (namely higher white blood cell count associated with higher neutrophil, lymphocyte, monocyte and/or eosinophil counts, shorter prothrombin time) were slight, not dose-related and/or with all the individual values close to or in the range of our historical background data. Consequently they were considered to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
When compared to controls, slightly lower inorganic phosphorus and protein levels were observed in animals given 600 mg/kg/day and slightly higher ALAT activity was noted in males given 150 mg/kg/day and in animals given 600 mg/kg/day. These changes were considered to be related to treatment with the test item.

The other changes observed (namely higher chloride and triglycerides levels, higher albumin/globulin ratio, lower cholesterol levels) were slight, not dose-related, without the same trend in the two sexes and/or with all the individual values close to or in the range of our historical background data. Consequently, they were considered to be of no toxicological significance.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Decreased tail pinch response was observed in 2/5, 1/5 and 1/5 males given 50, 150 and 600 mg/kg.day, respectively. As it was not dose-related, without the same trend in both sexes and as no other changes were seen in these animals, a relationship to treatment is considered to be doubtful.
There were no relevant differences in motor activity between control and treated animals.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The lower adrenal weights of the treated males and the higher adrenal wieghts of the treated females were dose-related. However, the differences were of opposite trend between the two sexes and microscopic examinations failed to reveal any changes that corresponded to this apparent. Consequently, this was considered to be of no toxicological importance.

Some other differences were observed in the mean weights of other organs, particularly the kidneys, the liver, the thymus and the thyroids. These differences were either not dose-related or minor and/or without the same trend in the two sexes. Moreover, the individual values of organ weights in the treated groups were generally in the range of the control group. The differences of the mean weights of these organs were thus considered to be of no toxicological importance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The findings wich were considered to be treatement-related were seen in the stomach, as follows:
- thickened and/or reddish mucosa, sometimes with depressed areas, in 4/5 males given 600 mg/kg/day and in 1/5 males given 150 mg/kg/day
- mucosa with several reddish and depressed foci in 2/5 females given 600 mg/kg/day
- external surface with several reddish areas in 1/5 females given 600 mg/kg/day.
These findings were shown to be treatment-related on microscopic examination.

The few other necropsy findings observed were those which are commonly recorded spontaneously in the untreated laboratory rat of this strain and age and thus were considered to be without toxicological significance.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Higher incidence of slight inflammatory changes was noted in the stomach of the animals given 600 mg/kg/day when compared with the control and other treated groups. This was characterized by moderate to slight submucosal edema and inflammatory cell infiltration. The pattern of the higher incidence of the slight inflammatory changes in the stomach was considered to be related to treatment at the 600 mgkg/day dose-level.

Apart from this changes in the stomach, the remaining entities reported were recognized as those that occur commonly in rats of this strain and age maintained in this laboratory. They were present with approximately equal incidence and degree of severity in both controls and treated animals and were consequently considered to be of no importance in a study of this nature.

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
clinical biochemistry
histopathology: non-neoplastic
Dose descriptor:
LOAEL
Effect level:
600 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The test item, VDPA Tetra Sodium Salt, when administered daily by gavage to rats at the dose-levels of 50, 150 and 600 mg/kg/day for 4 weeks was well tolerated up to 50 mg/kg/day.
At 150 and 600 mg/kg/day, it induced ptyalism, lower body weight gain, changes in biochemical parameters and microscopic lesions of the stomach. Microscopic examination showed evidence of inflammation in the stomach of the animals given 600 mg/kg/day.
Consequently, under the experimental conditions of the study, the NOEL was established at 50 mg/kg/day and the NOAEL was established at 150 mg/kg/day.
Executive summary:

The objective of the study was to evaluate the potential toxicity of the test item VDPA Tetra Sodium Salt, following daily oral administration (gavage) to rats for 4 weeks following the EU B.7 Guideline under GLP conditions.

Three treated groups of five male and five female rats received the test item, VDPA Tetra Sodium Salt, by gavage at the dose-levels of 50, 150 or 600 mg/kg/day for 4 weeks. An additional group of five males and five females received the vehicle alone (purified water) under the same experimental conditions and acted as a control group.

The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded once a week during the study. Detailed clinical observations were made before the beginning of the study and then once a week. Neurotoxic evaluation using functional observation battery (FOB) was carried out at the end of the treatment period as well as hematology and blood biochemical investigations.

On completion of the treatment period, the animals were killed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on selected tissues from animals.

No mortality occured during the study. There were no treatment-related changes in autonomic, physiological or neurotoxicological parameters, in motor acivity, in food consumption, in hematological parameters and in organ weights.

Ptyalism was observed in animals given 600 mg/kg/day. In addition signs of poor clinical condition (piloerection and round back) were observed in 1/5 females given 600 mg/kg/day. Slightly lower mean body weight gain was observed in females given 150 and 600 mg/kg/day. When compared to controls, slightly lower inorganic phosphorus and protein levels were observed in animals given 600 mg/kg/day and slightly higher ALAT activity was noted in males given 150 mg/kg/day and in animals given 600 mg/kg/day. Thickened and/or reddish mucosa in the stomach, sometimes with depressed areas or foci, or external surface with several reddish areas, were observed in animals given 150 and 600 mg/kg/day. Higher incidence of slight inflammatory changes was noted in the stomach of the animals given 600 mg/kg/day.

The test item, VDPA Tetra Sodium Salt, when administered daily by gavage to rats at the dose-levels of 50, 150 and 600 mg/kg/day for 4 weeks was well tolerated up to 50 mg/kg/day.

At 150 and 600 mg/kg/day, it induced ptyalism, changes in biochemical parameters and microscopic lesions of the stomach. Microscopic examination showed evidence of inflammation in the stomach of the animals given 600 mg/kg/day.

Consequently, under the experimental conditions of the study, the NOEL was established at 50 mg/kg/day and the NOAEL was established at 150 mg/kg/day, based on local effects on the stomach mucosa (reddish areas). However, the substance is known to have an extreme pH (11.7) which may cause a direct local effect on the acidic stomach environment, additionnally the body weight gain and food consumption during the study did no show any significant treatment-related effects.