Phosphonic acid, monoethyl ester, sodium salt (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 Jan - 01 Jun 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

(adopted 1995)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

(adopted 1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Republique Francaise, Premier Ministre, Groupe Interministeriel Des Produits Chimiques, Paris Cedex, France

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-960-2

EC Name:

-

Cas Number:

39148-16-8

Molecular formula:

C2H6O3P.Na

IUPAC Name:

sodium ethyl phosphonate

Test animals

Species:

rat

Strain:

other: Wistar (AF) RJ: WI (IOPS AF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 7 weeks
- Weight at study initiation: 223 - 281 g (males), 161 - 206 g (females)
- Fasting period before study: animals were fasted overnight for diet only before blood sampling and for diet and water before urine sampling
- Housing: individual in suspended stainless steel, wire mesh cages
- Diet: certified and irradiated rodent pellet diet "A04C" (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France), ad libitum
- Water: filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15 (average, not monitored)
- Photoperiod (hrs dark / hrs light): (12 / 12)

IN-LIFE DATES: From: 30 Jan To: 01 Mar 2001

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

not specified

Vehicle:

water

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was sufficiently soluble in water.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dosing solutions were sent for analysis to RCC Ltd, Environmental Chemistry and Pharmanalytics Division, Switzerland. Homogeinity of the test substance was verified at the lowest and highest doses. Concentrations of the test item in the vehicle were verified for each dose tested on the three first preparations. Stability of the test substance was demonstrated during the study at the lowest and highest dose.

Results of analysis
The results obtained for homogeneity, stability and concentration were within the expected ranges except the lowest concentration of the first preparation which was 111% of the nominal concentration (e.g. test 1: homogeneity: 91 - 98 and 103 - 105% of nominal concentration for 0.2 and 200 g/L, respectively; concentration: 95 and 104% of nominal concentration for 0.2 and 200 g/L, respectively; stability: 90 -95 and 92 - 104% of nominal concentration for 0.2 and 200 g/L, respectively; test 2: homogeneity: 111.2 - 112.5 and 90 - 96.2% of nominal concentration for 20 and 200 g/L, respectively; concentration: 90.4 - 111 and 93 - 102% of nominal concentration for 20 and 200 g/L, respectively).

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

(adjusted for purity)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

(adjusted for purity)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

(adjusted for purity)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of an acute oral study
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: animals were fasted overnight
- Rationale for selecting satellite groups: no satellite groups included

Positive control:

not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked for ill-health including blood or loose feces were included. Detailed physical examinations were performed at least weekly during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice during the acclimatisation period, on the first day of dosing and at weekly intervals thereafter and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the acclimatisation period and during week 4
- Dose groups that were examined: control and high-dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once during the study on Day 24 or 25
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all survivng animals
- Parameters checked: red blood cell count (RBC), haemoglobin, haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC) and differential count evaluation and platelet count (PLT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once during the study on Day 24 or 25
- Animals fasted: Yes
- How many animals: all suriving animals
- Parameters checked: total bilirubin (TBIL), glucose (GLUC), urea, creatinine (CREA), total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations, and aspartate aminotransferase, alanine aminotransferase (ALAT), alkaline phosphatase (AP) and gamma-glutamyltransferase (GGT) activities were assayed on plasma samples, total protein and albumin concentrations

URINALYSIS: Yes
- Time schedule for collection of urine: on Days 29, 30 or 31, prior to sacrifice
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: volume, pH, urinary refractive index (RI), glucose (GLU), bilirubin (BIL), ketone bodies (KET), occult blood (OCBL), protein (PRO) and urobilinogen (UOR), microscopic examination of the urinary sediment ( presence of red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the acclimatisation period and during week 4
- Functions tested: Grasping reflex, righting reflex, corneal reflex, pupillary reflex, auditory reflex and head shaking reflex

Sacrifice and pathology:

SACRIFICE: All surviving animals from all dose groups were sacrificed on study Days 29, 30 or 31. An approx. equal number of animals was randomly distributed amongst all groups.

GROSS PATHOLOGY: Yes
- Organs weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate, spleen, testes, thymus, thyroid (with parathyroid) and uterus were weighed fresh at scheduled sacrifice only. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
- Dose groups: all animals of the control and high-dose group, all decedents in the low- and mid-dose groups (lung, liver, thyroid gland,kidney and all macroscopic findings were also examined in the low- and mid-dose groups)

The following organs were sampled for pathological examinations:
Adrenal gland, Aorta, Articular surface (femoro-tibial), Bone (sternum), Bone marrow (sternum), Brain, Epididymis, Esophagus, Eye and optic nerve, Exorbital (lachrymal) gland, Harderian gland, Heart, Intestine (duodenum, jejunum, ileum,cecum, colon, rectum), Kidney, Larynx, Liver, Lung, Lymph nodes (submaxillary, mesenteric), Mammary gland, Ovary, Pancreas, Pituitary gland,Prostate, Sciatic nerve, Seminal vesicle, Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Submaxillary (salivary) gland, Testis,Thymus,Thyroid (with parathyroid), Tongue, Trachea, Urinary bladder, Uterus (including cervix), Vagina

Statistics:

Means and standard deviations were calculated for the analysed parameters for each sex separately for each group at each time period. All tests were performed at 1 and 5% significance levels. Statistical analysis were performed using ANOVA. Homogeinity of variance assumption was examined by Levene´s Test. If Levene's test indicated lack of homogeneity of variance (p<0.05), robust linear regression methods were used to test all treatment effects. The robust regression methods use variance estimators that make no assumptions regarding homogeneity of variance or normality of the data. They were used to test for overall treatment group differences (Wald chi-square tests), followed by individual Mests for exposed vs. control group comparisons when the overall treatment effect was significant. If Levene's test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS® 6.12 was used to evaluate the overall effect of treatment and, when a significant treatment effect was present, to compare each exposed group to control via Dunnett's test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 and 300 mg/kg bw/day, two and one animals, respectively, showed ocular discharge. At 300 mg/kg bw/day, two and one animals showed nasal discharge and abnormal dentition, respectively. However, since there was no dose-response relationship, the effects were considered incidental.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1000 mg/kg bw/day: one female died
300 mg/kg bw/day: one female died

The two females from the 300 and 1000 mg/kg bw/day group died during anesthesia for blood sampling on Day 24 and 25, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects on body weight were observed and the weight gain was comparable among the groups (mean weight gain weeks 0 -4: males: 148.9 ± 20.5, 142.0 ± 21.9, 147.5 ± 18.9 and 137.6 ± 14.0; females: 54.3 ± 10.9, 56.3 ± 8.2, 55.6 ± 13.5 and 58.7 ± 5.8 for control, low-, mid- and high-dose animals, respectively).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by the test substance (mean food consumption weeks 0 - 4: males: 27.7 ± 1.7, 27.4 ± 1.3, 27.8 ± 1.8 and 27.1 ± 1.4; females: 19.6 ± 1.6, 19.1 ± 1.5, 19.5 ± 1.5 and 19.3 ± 0.7 for control, low-, mid- and high-dose animals, respectively).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related abnormalities at the ophthalmological examination.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

100 and 300 mg/kg bw/day: decreased RBC in males

When compared with the control group, there were no treatment-related changes in hematological parameters in either sex. The lower RBC observed in males at 300 and 100 mg/kg/day (8.697 ± 0.302, 8.293* ± 0.329, 8.318* ± 0.257 and 8.469 ± 0.345 for control, low-, mid- and high-dose group, respectively) were considered to be of no toxicological significance because this change was slight and not seen at 1000 mg/kg bw/day and the RBC level was comparable in females (8.301 ± 0.318, 8.313 ± 0.569, 8.031 ± 0.527 and 8.226 ± 0.381 for control, low-, mid- and high-dose group, respectively). The other examined parameter did not reveal treatment-related changes and were comparable among the groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: decreased Cl (males)
300 mg/kg bw/day: decreased ASAT (males) and decreased Na (females)

When compared with the control group, there were no treatment-related changes in clinical chemistry parameters in either sex. Any statistical significant differences were considered to be of no toxicological significance because they were either not dose-related or the values were within the normal range of historical control (please refer to table 1).

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day: males showed decreased urinary pH values and a lower number of crystals (non adverse)

Males of the mid- and high-dose group revealed a statistically reduced mean urinary pH (males: 6.72 ± 0.36, 6.67 ± 0.25, 6.33 ± 0.25* and 5.83 ± 0.35***; females: 5.8 ± 0.42, 5.83 ± 0.35, 5.44 ± 0.39 and 5.38 ± 0.35 for control, low-, mid- and high-dose group, respectively). Furthermore, a lower number of crystals was determined in mid- and high-dose males (please refer to Table 2). As these changes were only slight and not linked to alterations in clinical or histopathological parameter, they are not considered as adverse.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The neurotoxicity assessment did not reveal any effects on neurobehavior.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: increased relative liver weight in males, increased absolute brain weight in females (non adverse)

Low-dose males revealed a statistically significant higher relative liver weight whereas the absolute liver weight and the liver-brain/weight ratio remained unaffected. Furthermore, the absolute brain weight of low-dose females was statistically significant increased without altering the relative brain weight (please refer to Table 3). In the absence of a dose-relationship, the effects on organ weights in low-dose animals were considered as fortuitous and not considered as adverse. The weights of the remaining organs (heart, pituitary gland, spleen, kidneys, adrenal glands, thymus, thyroid gland, epididymis, prostate, testes, uterus and ovaries) were comparable among the dose groups in both genders.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

all groups: abnormalities in harderian gland, kidneys, liver, lung, spleen or uterus (non adverse)

Scheduled sacrifices: In general, most animals from control and test groups did not show abnormalities in gross pathology (males: 6, 7, 5 and 6; females: 9, 6, 2 and 8 animals without abnormalities in control, low-, mid- and high-dose groups). Changes observed on harderian gland, kidney, liver, lung, spleen, eyes or uterus were observed in only few animals without a dose-relationship and are therefore considered as incidental in origin and unrelated to treatment (please refer to Table 4).

Unscheduled death: The deceased from the high-dose group showed red foci in the thymus and adrenal gland. Moreover, the liver appeared dark. All organs from the deceased of the mid-dose group appeared normal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

High-dose males revealed a higher incidence of slight vacuolation in the zona fasciculata of the adrenal gland (2/10 vs 5/10 in control and high-dose males). As the effect was not observed in any female, toxicological significance of this effect is questionable. Moreover, 2/10 high-dose males showed signs of slight, unilateral, focal atrophy of the retina, which was also determined in 1/3 mid-dose females. In the kidney, increased incidences of focal basophilic tubulus and mineralisation of the inner medulla was observed in males (1/10, 1/10, 3/10 and 5/9; 0/10, 0/10, 0/10 and 3/9 of the control, low-, mid-and high-dose group, respectively). Furthermore, male animals showed increasing incidences for the following abnormalities of the lung: mononuclear inflammation (5/10, 9/10, 9/10 and 7/10 for control, low-, mid- and high-dose animals) and chronic interstitial inflammation (3/10 for control and 6/10 for each dose group). As the effects observed are only present in one sex and no clear dose-response was present, biological relevance remains questionable. Further abnormalities observed in histopathology were comparable among the groups and are therefore considered as incidental in origin and unrelated to treatment (please refer to Table 5).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Male animals showed increasing incidences for the following abnormalities of the lung: hyperplasia of regenerative type II pneumocytes (2/10, 4/10, 3/10 and 4/10 for control, low-, mid- and high-dose animals) and slight, bronchiolar regenerative hyperplasia (0/10, 1/10, 0/10 and 2/10 for control, low-, mid- and high-dose animals). Female test animals showed a higher incidence of diffuse, epithelial hyperplasia in the high-dose group (1/10 vs 3/9 for control and high-dose group) in the trachea. As the effects observed are only present in one sex and no clear dose-response was present, biological relevance remains questionable. Further abnormalities observed in histopathology were comparable among the groups and are therefore considered as incidental in origin and unrelated to treatment (please refer to Table 5).

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed on the examined parameters (applied concentrations were adjusted for purity).

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In this 28-day repeated dose toxicity study performed according to OECD 407 and in compliance with GLP, the NOAEL for both sexes was greater than 1000 mg/kg bw/day.