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Description of key information

A 3-months feeding study in rats with technical tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate (50, 200, 1000, 5000 ppm = male rats: 0, 4.17, 14.91, 84.11, 424.41 mg/kg bw/day, female rats: 0, 6.05, 12.51, 125.48, 632.65 mg/kg bw/day ) results in a NOAEL equal or higher than 5000 ppm (equivalent to about 424 mg/kg bw/day  for male rats and 632 mg/kg bw/day for female rats). Conclusion for subchronic exposure: Low toxicity, no damage in oral doses up to 424 mg/kg/body weight/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not indicated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The test substance is not fully specified on composition and purity and no analytical verification of the test substance was performed. Nevertheless, as the performance of study was scientifically-toxicologically correct (and a purity lower than 100% would most likely increase the NOAEL), reliability 2 was assigned.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Principles of method if other than guideline:
The OECD original guideline 408 was adopted in 1981, the current guideline was adopted in 1998, but the study was performed in 1976.
Maybe for this reason,
- physical nature, purity and physico-chemical properties of the test substance are not explicitly indicated
- identification data of the test substance is not indicated
- no analytical verification of the test substance was performed
- ophthalmological examinations are not (explicitly) cited.
- sensory tests of reactivity to stimuli are not (explicitly) cited.
A dose of 0, 50, 200, 1000, or 5000 ppm corresponds with an ingestion of the test substance for male rats of 0, 4.17, 14.91, 84.11, or 424.41 mg/kg bw/day) and for female rats of 0, 6.05, 12.51, 125.48 or 632.65 mg/kg bw/day)
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- SPF
- Source: Winkelmann, Kirchborchen, Germany
- Age at study initiation: 28-32 days
- Weight at study initiation (male rats): Average 80-85 g
- Weight at study initiation (female rats): Average 78-81 g
- Housing: cages Makrolon, type II; 1 animals per cage
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/-1°C


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
NA
Duration of treatment / exposure:
3 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
50 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
200 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
5000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
test group: 15 animals / sex/ dose level
control group: 30 animals / sex
Control animals:
yes, plain diet
Details on study design:
OBSERVATIONS:
- The animals were observed for changes and symptoms daily.

BODY WEIGHT AND FOOD/WATER CONSUMPTION:
- The weekly food consumption was calculated by weighing the feed before and after one week of consumption.
- The bodyweights of all involved animals were recorded weekly.

EXAMINATIONS:
- The clinical investigations were undertaken with 5 male and 5 female rats per dose level one and three months after start of this the feeding study.
Haematology:
- Determination of haemoglobin concentration according to the cyan-methemoglobin method (VAN KAMPEN and ZIJLSTRA, 1961)
- Determination of haematocrit (microhematocrit method by centrifugation)
- Counting of erythrocytes and leucocyte by the Coulter counter, model ZF.
- Determination of mean corpuscular haemoglobin concentration (MCHC)
- Determination of mean corpuscular volume (MCV)
- Counting of reticulocytes (stained with cresyl brilliant blue)
- Counting of thrombocytes by the Coulter counter, model FN
- Assessment of the differential blood count (staining according to WRIGHT; 630-times magnified)
- Determination of the thromboplastine time (according to QUICK, 1951)
Liver function test:
The following enzymes were determined in the heparin plasma:
- alkaline phosphatase (according to HAUSAMEN et al., 1967)
- glutamic oxal acetic transaminase (GOT, according to KARMEN et al., 1955)
- glutamate-pyruvate transaminase (GPT according to WROBLEWSKI and LA DUE, 1955)
- bilirubin (according to JENDRASSIK and GROF, 1938)
- total protein (according to WEICHSELBAUM, 1946)
Urinalysis and kidney function test:
- 16h urine was sampled and analysed for glucose, haemoglobin, pH and bile colour by test stripes
- urine protein (according to RICHTERICH, 1968)
- microscopic investigations of urine sediment after centrifugation of the urine
- determination of urea (acc. FAWCETT and SCOTT, 1960) and creatinine (acc. POPPER et al., 1937)
Moreover, the electrolytes sodium, potassium and calcium were determined by flame photometry.
Blood sugar and cholesterol:
- glucose: enzymatic colorimetric with glucose-oxidase-peroxidase (acc. HUGGET and NIXON, 1957)
- cholesterol (acc. to WATSON, 1960)
Determination of sodium, ferrum, phosphorus, potassium and calcium in blood serum:
- sodium, potassium and calcium by flame photometry
- ferrum (acc. TRINDER, 1956)
- inorganic phosphorus (acc. RAABE, 1955)


PATHOLOGY:
The weights of the following organs of all animals were determined: thyroid, thymus, heart, lungs, liver, spleen, kidneys, adrenal glands, testes, and ovaries.

For histological examinations the organs of five male and five female rats were fixated, plastinated and stained:
Aorta, eyes, large and small intestines, brain, urinary bladder, heart, testes, hypotheses, liver, lungs, lymph nodes, stomach, spleen, A. quadriceps, epididymis, adrenal glands, kidneys, oesophagus, parotid gland, Os femoris, ovaries, pancreas, prostate, seminal vesicle, thyroid, sternum, thymus, trachea and uterine horn.
All these organs of animals that had received the highest dose (5000 ppm) were hispathologically examined.
The following organs of animals that had received doses of 50, 200, or 1000 ppm were also examined: Heart, testes, hypotheses, liver, spleen, epididymis, adrenal glands, kidneys, Os femoris, ovaries, pancreas, thyroid, sternum, thymus and uterine horn.
Positive control:
No positive control.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATION and DETAILED CLINICAL OBSERVATIONS: Yes
- The animals were observed for changes and symptoms daily (appearance, e.g. of fur, behaviour, e.g. concerning drinking and eating, motor activity ).

BODY WEIGHT: Yes
- The bodyweights of all involved animals were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was determined weekly. Based on that the mean daily consumption was calculated and given as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: One and three months after start of the feeding study
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 5 per sex and dose level
- Parameters examined: See "study design"

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: One and three months after start of the feeding study
- Animals fasted: No data
- How many animals: 5 per sex and dose level
- Parameters examined: See "study design

URINALYSIS: Yes
- Time schedule for collection of urine: Not indicated
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined: See "study design

NEUROBEHAVIOURAL EXAMINATION: No data
Statistics:
For statistical evaluation of the study results, the rank-sum test (distribution free) according to WILCOXON (see also WINNE) was applied.
The p values less than 0.05 were regarded as significant.
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 specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
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:
no effects observed
Details on results:
Based on the body weights at the middle of the study and the food consumption of one day, the 5000 ppm dose was equivalent to 424 mg test substance/ kg bw for male rats and 632 mg test substance / kg bw for female rats.
No test animal died over the whole test period.
Dose descriptor:
NOAEL
Effect level:
>= 5 000 ppm
Sex:
male/female
Basis for effect level:
other: highest dose level
Critical effects observed:
not specified

No remarks

Conclusions:
Doses of up to 5000 ppm tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate applied over 3 months were tolerated without any effects.
The NOAEL of the tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate is equal or higher than 5000 ppm (equivalent to about 424 mg/kg bw for male rats and 632 mg/kg bw for female rats).
Executive summary:

A 3-months feeding study was performed in 15 female and 15 male rats with technical tetrasodium hydrogen 2-phosphonatobutane-1,2,4-tricarboxylate (50, 200, 1000, 5000 ppm = male rats: 0, 4.17, 14.91, 84.11, 424.41 mg/kg bw/day, female rats: 0, 6.05, 12.51, 125.48, 632.65 mg/kg bw/day). The control animals (30 females, 30 males) received no test substance (0 ppm).

All doses were tolerated without any effects as could be shown by overall observations and examinations: Appearance, behaviour, development, and mortality as well as blood, blood glucose and cholesterol, metabolism of electrolytes (NA, K, Ca), Ferrum and Phosphorus were not affected by doses up to 5000 ppm. The same was proven for the kidneys by urinalyses, clinical chemistry and pathological and histopathological examinations. Gross necropsy and histological examinations did not reveal any adverse effects due to the test substance.

Thus, the NOAEL is equal or higher than 5000 ppm (equivalent to about 424 mg/kg bw for male rats and 632 mg/kg bw for female rats).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
424 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The materials/methods and results are described in detail und are sufficient for evaluation

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In an early and limited documented subchronic toxicity study technical tetrasodium hydrogen 2-phosphonatobutane-1,2,4- tricarboxylate was administered to 15 female and 15 male Wistar rats in diet, at dose levels of 0, 50, 200, 1000, and 5000 ppm.

All doses were tolerated without any effects as could be shown by overall observations and examinations: Appearance, behaviour, development, and mortality as well as blood, blood glucose and cholesterol, metabolism of electrolytes (Na, K, Ca), Ferrum and Phosphorus were not affected by doses up to 5000 ppm. The same was proven for the kidneys by urinalyses, clinical chemistry and pathological and histopathological examinations. Gross necropsy and histological examinations did not reveal any adverse effects due to the test substance.

Based on these result the highest dose level was chosen as NOAEL. Thus, the NOAEL is = 5000 ppm. 5000 ppm are equivalent to about 424 mg/kg bw/day for male rats and 632 mg/kg bw/day for female rats.

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study is used

Justification for classification or non-classification

Based on the (worst case) NOAEL of 424 mg/kg bw/day (which is >100 mg/kg bw/day and therefore exceeds the respective guidance value for classification), tetrasodium hydrogen 2-phosphonatobutane-tricarboxylate ("PBTCNa4") is not classified as according to EU Directive 67/548/EC and according to EU regulation No. 1272/2008 (GHS; amending and repealing Directives 67/548/EEC and 1999/45/EC, and amending Regulation (EC) No 1907/2006) as a substance that has produced significant toxicity in humans or that, on the basis of evidence from studies in experimental animals, can be presumed to have the potential to produce significant toxicity in humans following repeated exposure (GHS category 1) or a substance that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposure (GHS category 2).